Compositions and methods for the diagnosis and treatment of herpes simplex virus infection

ABSTRACT

Compounds and methods for the diagnosis and treatment of HSV infection are provided. The compounds comprise polypeptides that contain at least one antigenic portion of an HSV polypeptide and DNA sequences encoding such polypeptides. Pharmaceutical compositions and vaccines comprising such polypeptides or DNA sequences are also provided, together with antibodies directed against such polypeptides. Diagnostic kits are also provided comprising such polypeptides and/or DNA sequences and a suitable detection reagent for the detection of HSV infection in patients and in biological samples.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional Application No.60/277,438 filed Mar. 20, 2001 and U.S. Provisional Application No.60/215,458 filed Jun. 29, 2000 and are incorporated in their entirety byreference herein.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates generally to the detection andtreatment of HSV infection. In particular, the invention relates topolypeptides comprising HSV antigens, DNA encoding HSV antigens, and theuse of such compositions for the diagnosis and treatment of HSVinfection.

BACKGROUND OF THE INVENTION

[0003] The herpes viruses include the herpes simplex viruses (HSV),comprising two closely related variants designated types 1 (HSV-1) and 2(HSV-2). HSV is a prevalent cause of genital infection in humans, withan estimated annual incidence of 600,000 new cases and with 10 to 20million individuals experiencing symptomatic chronic recurrent disease.The asymptomatic subclinical infection rate may be even higher. Forexample, using a type-specific serological assay, 35% of an unselectedpopulation of women attending a health maintenance organization clinicin Atlanta had antibodies to HSV type 2 (HSV-2). Although continuousadministration of antiviral drugs such as acyclovir ameliorates theseverity of acute HSV disease and reduces the frequency and duration ofrecurrent episodes, such chemotherapeutic intervention does not abortthe establishment of latency nor does it alter the status of the latentvirus. As a consequence, the recurrent disease pattern is rapidlyreestablished upon cessation of drug treatment.

[0004] The genome of at least one strain of herpes simplex virus (HSV)has been characterized. It is approximately 150 kb and encodes about 85known genes, each of which encodes a protein in the range of 50-1000amino acids in length. Unknown, however, are the immunogenic portions,particularly immunogenic epitopes, that are capable of eliciting aneffective T cell immune response to viral infection.

[0005] Thus, it is a matter of great medical and scientific need toidentify immunogenic portions, preferably epitopes, of HSV polypeptidesthat are capable of eliciting an effective immune response to HSVinfection. Such information will lead to safer and more effectiveprophylactic pharmaceutical compositions, e.g., vaccine compositions, tosubstantially prevent HSV infections, and, where infection has alreadyoccurred, therapeutic compositions to combat the disease. The presentinvention fulfills these and other needs.

SUMMARY OF THE INVENTION

[0006] The present invention provides compositions and methods for thediagnosis and therapy of HSV infection. In one aspect, the presentinvention provides polypeptides comprising an immunogenic portion of aHSV antigen, or a variant or biological functional equivalent of such anantigen. Certain preferred portions and other variants are immunogenic,such that the ability of the portion or variant to react withantigen-specific antisera is not substantially diminished. Withincertain embodiments, the polypeptide comprises an amino acid sequenceencoded by a polynucleotide sequence selected from the group consistingof (a) a sequence of any one of SEQ ID NO: 1, 4, 8-9, 13, 16, 19, 24,35-38, 48-49, and 52-53; (b) a complement of said sequence; and (c)sequences that hybridize to a sequence of (a) or (b) under moderatelystringent conditions. In specific embodiments, the polypeptides of thepresent invention comprise at least a portion, preferably at least animmunogenic portion, of a HSV protein that comprises some or all of anamino acid sequence recited in any one of SEQ ID NO: 2, 3, 5, 6, 7,10-12, 14-15, 17-18, 20-23, 25-33, 39-47, 50-51, and 54-64, includingvariants and biological functional equivalents thereof.

[0007] The present invention further provides polynucleotides thatencode a polypeptide as described above, or a portion thereof (such as aportion encoding at least 15 contiguous amino acid residues of a HSVprotein), expression vectors comprising such polynucleotides and hostcells transformed or transfected with such expression vectors.

[0008] In a related aspect, polynucleotide sequences encoding the abovepolypeptides, recombinant expression vectors comprising one or more ofthese polynucleotide sequences and host cells transformed or transfectedwith such expression vectors are also provided.

[0009] In another aspect, the present invention provides fusion proteinscomprising one or more HSV polypeptides, for example in combination witha physiologically acceptable carrier or immunostimulant for use aspharmaceutical compositions and vaccines thereof.

[0010] The present invention further provides pharmaceuticalcompositions that comprise: (a) an antibody, either polyclonal andmonoclonal, or antigen-binding fragment thereof that specifically bindsto a HSV protein; and (b) a physiologically acceptable carrier.

[0011] Within other aspects, the present invention providespharmaceutical compositions that comprise one or more HSV polypeptidesor portions thereof disclosed herein, or a polynucleotide moleculeencoding such a polypeptide, and a physiologically acceptable carrier.The invention also provides vaccines for prophylactic and therapeuticpurposes comprising one or more of the disclosed polypeptides and animmunostimulant, as defined herein, as well as vaccines comprising oneor more polynucleotide sequences encoding such polypeptides and animmunostimulant.

[0012] In yet another aspect, methods are provided for inducingprotective immunity in a patient, comprising administering to a patientan effective amount of one or more of the above pharmaceuticalcompositions or vaccines. Any of the polypeptides identified for use inthe treatment of patients can be used in conjunction with pharmaceuticalagents used to treat herpes infections, such as, but not limited to,Zovirax® (Acyclovir), Valtrex® (Valacyclovir), and Famvir®(Famcyclovir).

[0013] In yet a further aspect, there are provided methods for treating,substantially preventing or otherwise ameliorating the effects of an HSVinfection in a patient, the methods comprising obtaining peripheralblood mononuclear cells (PBMC) from the patient, incubating the PBMCwith a polypeptide of the present invention (or a polynucleotide thatencodes such a polypeptide) to provide incubated T cells andadministering the incubated T cells to the patient. The presentinvention additionally provides methods for the treatment of HSVinfection that comprise incubating antigen presenting cells with apolypeptide of the present invention (or a polynucleotide that encodessuch a polypeptide) to provide incubated antigen presenting cells andadministering the incubated antigen presenting cells to the patient.Proliferated cells may, but need not, be cloned prior to administrationto the patient. In certain embodiments, the antigen presenting cells areselected from the group consisting of dendritic cells, macrophages,monocytes, B-cells, and fibroblasts. Compositions for the treatment ofHSV infection comprising T cells or antigen presenting cells that havebeen incubated with a polypeptide or polynucleotide of the presentinvention are also provided. Within related aspects, vaccines areprovided that comprise: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) an immunostimulant.

[0014] The present invention further provides, within other aspects,methods for removing HSV-infected cells from a biological sample,comprising contacting a biological sample with T cells that specificallyreact with a HSV protein, wherein the step of contacting is performedunder conditions and for a time sufficient to permit the removal ofcells expressing the protein from the sample.

[0015] Within related aspects, methods are provided for inhibiting thedevelopment of HSV infection in a patient, comprising administering to apatient a biological sample treated as described above. In furtheraspects of the subject invention, methods and diagnostic kits areprovided for detecting HSV infection in a patient. In one embodiment,the method comprises: (a) contacting a biological sample with at leastone of the polypeptides or fusion proteins disclosed herein; and (b)detecting in the sample the presence of binding agents that bind to thepolypeptide or fusion protein, thereby detecting HSV infection in thebiological sample. Suitable biological samples include whole blood,sputum, serum, plasma, saliva, cerebrospinal fluid and urine. In oneembodiment, the diagnostic kits comprise one or more of the polypeptidesor fusion proteins disclosed herein in combination with a detectionreagent. In yet another embodiment, the diagnostic kits comprise eithera monoclonal antibody or a polyclonal antibody that binds with apolypeptide of the present invention.

[0016] The present invention also provides methods for detecting HSVinfection comprising: (a) obtaining a biological sample from a patient;(b) contacting the sample with at least two oligonucleotide primers in apolymerase chain reaction, at least one of the oligonucleotide primersbeing specific for a polynucleotide sequence disclosed herein; and (c)detecting in the sample a polynucleotide sequence that amplifies in thepresence of the oligonucleotide primers. In one embodiment, theoligonucleotide primer comprises at about 10 contiguous nucleotides of apolynucleotide sequence peptide disclosed herein, or of a sequence thathybridizes thereto.

[0017] In a further aspect, the present invention provides a method fordetecting HSV infection in a patient comprising: (a) obtaining abiological sample from the patient; (b) contacting the sample with anoligonucleotide probe specific for a polynucleotide sequence disclosedherein; and (c) detecting in the sample a polynucleotide sequence thathybridizes to the oligonucleotide probe. In one embodiment, theoligonucleotide probe comprises at least about 15 contiguous nucleotidesof a polynucleotide sequence disclosed herein, or a sequence thathybridizes thereto.

[0018] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description. Allreferences disclosed herein are hereby incorporated by reference intheir entirety as if each was incorporated individually.

[0019] Sequence Identifiers

[0020] SEQ ID NO: 1 sets forth a polynucleotide sequence of an isolatedclone designated HSV2I_UL39fragH12A12;

[0021] SEQ ID NO: 2 sets forth an amino acid sequence, designatedH12A12orf1.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 1;

[0022] SEQ ID NO: 3 sets forth the amino acid sequence of the fulllength HSV-2 UL39 protein;

[0023] SEQ ID NO: 4 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_US8AfragD6.B_B11_T7Trc.seq;

[0024] SEQ ID NO: 5 sets forth an amino acid sequence, designatedD6Borf1.pro, of an open reading frame encoded within the polynucleotideof SEQ ID NO: 4;

[0025] SEQ ID NO: 6 sets forth an amino acid sequence, designatedD6Borf2.pro, of an open reading frame encoded within the polynucleotideof SEQ ID NO: 4;

[0026] SEQ ID NO: 7 sets forth the amino acid sequence of the fulllength HSV-2 US8A protein;

[0027] SEQ ID NO: 8 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_US4fragF10B3_T7Trc.seq;

[0028] SEQ ID NO: 9 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_US3fragF10B3_T7P.seq;

[0029] SEQ ID NO: 10 sets forth an amino acid sequence, designatedF10B3orf2.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 8;

[0030] SEQ ID NO: 11 sets forth an amino acid sequence, designated8F10B3orf1.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 9;

[0031] SEQ ID NO: 12 sets forth the amino acid sequence of the fulllength HSV-2 US3 protein;

[0032] SEQ ID NO: 13 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_UL46fragF11F5_T7Trc.seq

[0033] SEQ ID NO: 14 sets forth an amino acid sequence, designatedF11F5orf1.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 13;

[0034] SEQ ID NO: 15 sets forth the amino acid sequence of the fulllength HSV-2 UL46 protein;

[0035] SEQ ID NO: 16 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_UL27fragH2C7_T7Trc.seq

[0036] SEQ ID NO: 17 sets forth an amino acid sequence, designatedH2C7orf1.pro, of an open reading frame encoded within the polynucleotideof SEQ ID NO: 16;

[0037] SEQ ID NO: 18 sets forth the amino acid sequence of the fulllength HSV-2 UL27 protein;

[0038] SEQ ID NO: 19 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_UL18fragF10A1_rc.seq;

[0039] SEQ ID NO: 20 sets forth an amino acid sequence, designatedF10A1orf3.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 19;

[0040] SEQ ID NO: 21 sets forth an amino acid sequence, designatedF10A1orf2.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 19;

[0041] SEQ ID NO: 22 sets forth an amino acid sequence, designatedF10A1orf1.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 19;

[0042] SEQ ID NO: 23 sets forth the amino acid sequence of the fulllength HSV-2 UL18 protein;

[0043] SEQ ID NO: 24 sets forth a polynucleotide sequence of an isolatedclone designated HSV2II_UL15fragF10A12_rc.seq;

[0044] SEQ ID NO: 25 sets forth an amino acid sequence, designatedF10A12orf1.pro, of an open reading frame encoded within thepolynucleotide of SEQ ID NO: 24;

[0045] SEQ ID NO: 26 sets forth the amino acid sequence of the fulllength HSV-2 UL15 protein;

[0046] SEQ ID NO:27 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL46 gene;

[0047] SEQ ID NO:28 sets forth the amino acid sequence of a 15-merpoly-eptide derived from an immunogenic portion of the HSVII UL46 gene;

[0048] SEQ ID NO:29 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL46 gene;

[0049] SEQ ID NO:30 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL46 gene;

[0050] SEQ ID NO:31 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL46 gene;

[0051] SEQ ID NO:32 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL18 gene;

[0052] SEQ ID NO:33 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL18 gene;

[0053] SEQ ID NO:34 sets forth a nucleotide sequence of an isolatedclone designated RL2_E9A4_5_consensus.seq;

[0054] SEQ ID NO:35 sets forth the nucleotide sequence of the fulllength HSV-2 RL2 gene;

[0055] SEQ ID NO:36 sets for the nucleotide sequence of an isolatedclone designated UL23_(—)22_C12A12_consensus.seq;

[0056] SEQ ID NO:37 sets forth the nucleotide sequence of the fulllength HSV-2 UL23 protein;

[0057] SEQ ID NO:38 sets forth the nucleotide sequence of the fulllength HSV-2 UL22 protein;

[0058] SEQ ID NO:39 sets forth an amino acid sequence, designatedHSV2_UL23, of an open reading frame encoded by the polynucleotide of SEQID NO: 37;

[0059] SEQ ID NO:40 sets forth an amino acid sequence designatedHSV2_UL23 of an open reading frame encoded within the polynucleotides ofSEQ ID NO:36;

[0060] SEQ ID NO:41 sets forth an amino acid sequence designatedHSV2_UL22 of an open reading frame encoded within the polynucleotides ofSEQ ID NO:36;

[0061] SEQ ID NO:42 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL23 gene;

[0062] SEQ ID NO:43 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL23 gene;

[0063] SEQ ID NO:44 sets forth the amino acid sequence of a 15-merpolypeptide derived from an immunogenic portion of the HSVII UL23 gene;

[0064] SEQ ID NO:45 sets forth an amino acid sequence, designatedHSV2_UL22, of an open reading frame encoded by the polynucleotide of SEQID NO :38;

[0065] SEQ ID NO:46 sets forth an amino acid sequence, designatedRL2_E9A4_5_consensus.seq, of an open reading frame encoded by thepolynucleotide of SEQ ID NO:34;

[0066] SEQ ID NO:47 sets forth an amino acid sequence, designatedHSV2_RL2, of an open reading frame encoded by the polynucleotide of SEQID NO:35;

[0067] SEQ ID NO:48 sets forth a nucleotide sequence of an isolatedclone designated G10_UL37consensus.seq;

[0068] SEQ ID NO:49 sets forth the nucleotide sequence of the fulllength HSV-2 UL37 gene;

[0069] SEQ ID NO:50 sets forth an amino acid sequence, designatedHSV2_UL37, of an open reading frame encoded by the polynucleotide of SEQID NO:48; and

[0070] SEQ ID NO:51 sets forth an amino acid sequence, designatedHSV2_UL37, of an open reading frame encoded by the polynucleotide of SEQID NO:49;

[0071] SEQ ID NO:52 sets forth the DNA sequence derived from the insertof clone UL46fragF11F5;

[0072] SEQ ID NO:53 sets forth the DNA sequence derived from the insertof clone G10;

[0073] SEQ ID NO:54 sets forth the amino acid sequence derived from theinsert of clone UL46fragF11F5;

[0074] SEQ ID NO:55 sets forth the amino acid sequence derived from theinsert of clone G10;

[0075] SEQ ID NO:56 is amino acid sequence of peptide #23 (amino acids688-702) of the HSV-2 gene UL15;

[0076] SEQ ID NO:57 is amino acid sequence of peptide #30 (amino acids716-730) of the HSV-2 gene UL15;

[0077] SEQ ID NO:58 is amino acid sequence of peptide #7 (amino acids265-279) of the HSV-2 gene UL23;

[0078] SEQ ID NO:59 is amino acid sequence of peptide #2 (amino acids621-635) of the HSV-2 gene UL46;

[0079] SEQ ID NO:60 is amino acid sequence of peptide #8 (amino acids645-659) of the HSV-2 gene UL46;

[0080] SEQ ID NO:61 is amino acid sequence of peptide #9 (amino acids649-663) of the HSV-2 gene UL46;

[0081] SEQ ID NO:62 is amino acid sequence of peptide #11 (amino acids657-671) of the HSV-2 gene UL46;

[0082] SEQ ID NO:63 is amino acid sequence of peptide #33 (amino acids262-276) of the HSV-2 gene US3;

[0083] SEQ ID NO:64 is amino acid sequence of peptide #5 (amino acids99-113) of the HSV-2 gene US8A.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0084] As noted above, the present invention is generally directed tocompositions and methods for making and using the compositions,particularly in the therapy and diagnosis of HSV infection. Certainillustrative compositions described herein include HSV polypeptides,polynucleotides encoding such polypeptides, binding agents such asantibodies, antigen presenting cells (APCs) and/or immune system cells(e.g., T cells). Certain HSV proteins and immunogenic portions thereofcomprise HSV polypeptides that react detectably (within an immunoassay,such as an ELISA or Western blot) with antisera of a patient infectedwith HSV.

[0085] Therefore, the present invention provides illustrativepolynucleotide compositions, illustrative polypeptide compositions,immunogenic portions of said polynucleotide and polypeptidecompositions, antibody compositions capable of binding suchpolypeptides, and numerous additional embodiments employing suchcompositions, for example in the detection, diagnosis and/or therapy ofhuman HSV infections.

[0086] Polynucleotide Compositions

[0087] As used herein, the terms “DNA segment” and “polynucleotide”refer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

[0088] As will be understood by those skilled in the art, the DNAsegments of this invention can include genomic sequences, extra-genomicand plasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

[0089] “Isolated,” as used herein, means that a polynucleotide issubstantially away from other coding sequences, and that the DNA segmentdoes not contain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. Of course, this refers to the DNA segment as originallyisolated, and does not exclude genes or coding regions later added tothe segment by the hand of man.

[0090] As will be recognized by the skilled artisan, polynucleotides maybe single-stranded (coding or antisense) or double-stranded, and may beDNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

[0091] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes an HSV protein or a portion thereof) ormay comprise a variant, or a biological or antigenic functionalequivalent of such a sequence. Polynucleotide variants may contain oneor more substitutions, additions, deletions and/or insertions, asfurther described below, preferably such that the immunogenicity of theencoded polypeptide is not diminished, relative to a native HSV protein.The effect on the immunogenicity of the encoded polypeptide maygenerally be assessed as described herein. The term “variants” alsoencompasses homologous genes of xenogenic origin.

[0092] When comparing polynucleotide or polypeptide sequences, twosequences are said to be “identical” if the sequence of nucleotides oramino acids in the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

[0093] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0094] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85: 2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0095] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. MolBiol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. In oneillustrative example, cumulative scores can be calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix can beused to calculate the cumulative score. Extension of the word hits ineach direction are halted when: the cumulative alignment score falls offby the quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.

[0096] Preferably, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

[0097] Therefore, the present invention encompasses polynucleotide andpolypeptide sequences having substantial identity to the sequencesdisclosed herein, for example those comprising at least 50% sequenceidentity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide or polypeptide sequence of this invention using themethods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

[0098] In additional embodiments, the present invention providesisolated polynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

[0099] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. For example, illustrative DNAsegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

[0100] In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5× SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5× SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2× SSC containing 0.1% SDS.

[0101] Moreover, it will be appreciated by those of ordinary skill inthe art that, as a result of the degeneracy of the genetic code, thereare many nucleotide sequences that encode a polypeptide as describedherein. Some of these polynucleotides bear minimal homology to thenucleotide sequence of any native gene. Nonetheless, polynucleotidesthat vary due to differences in codon usage are specificallycontemplated by the present invention. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

[0102] Probes and Primers

[0103] In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 nucleotide long contiguous sequence that has the same sequence as, oris complementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

[0104] The ability of such nucleic acid probes to specifically hybridizeto a sequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

[0105] Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

[0106] The use of a hybridization probe of about 15-25 nucleotides inlength allows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

[0107] Hybridization probes may be selected from any portion of any ofthe sequences disclosed herein. All that is required is to review thesequence set forth in the sequences disclosed herein, or to anycontinuous portion of the sequence, from about 15-25 nucleotides inlength up to and including the full length sequence, that one wishes toutilize as a probe or primer. The choice of probe and primer sequencesmay be governed by various factors. For example, one may wish to employprimers from towards the termini of the total sequence.

[0108] Small polynucleotide segments or fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCRTM technology of U.S. Pat.No. 4,683,202 (incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

[0109] The nucleotide sequences of the invention may be used for theirability to selectively form duplex molecules with complementarystretches of the entire gene or gene fragments of interest. Depending onthe application envisioned, one will typically desire to employ varyingconditions of hybridization to achieve varying degrees of selectivity ofprobe towards target sequence. For applications requiring highselectivity, one will typically desire to employ relatively stringentconditions to form the hybrids, e.g., one will select relatively lowsalt and/or high temperature conditions, such as provided by a saltconcentration of from about 0.02 M to about 0.15 M salt at temperaturesof from about 50° C. to about 70° C. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating relatedsequences.

[0110] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0111] Polynucleotide Identification and Characterization

[0112] Polynucleotides may be identified, prepared and/or manipulatedusing any of a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for HSV-associated expression (i.e.,expression that is at least two fold greater in infected versus normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using a Synteni microarray(Palo Alto, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA94:2150-2155, 1997). Alternatively, polynucleotides may be amplifiedfrom cDNA prepared from cells expressing the proteins described herein.Such polynucleotides may be amplified via polymerase chain reaction(PCR). For this approach, sequence-specific primers may be designedbased on the sequences provided herein, and may be purchased orsynthesized.

[0113] An amplified portion of a polynucleotide of the present inventionmay be used to isolate a full length gene from a suitable library (e.g.,an HSV cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

[0114] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

[0115] Alternatively, there are numerous amplification techniques forobtaining a full length coding sequence from a partial cDNA sequence.Within such techniques, amplification is generally performed via PCR.Any of a variety of commercially available kits may be used to performthe amplification step. Primers may be designed using, for example,software well known in the art. Primers are preferably 22-30 nucleotidesin length, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

[0116] One such amplification technique is inverse PCR (see Triglia etal., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60, 1991). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

[0117] In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

[0118] Polynucleotide Expression in Host Cells

[0119] In other embodiments of the invention, polynucleotide sequencesor fragments thereof which encode polypeptides of the invention, orfusion proteins or functional equivalents thereof, may be used inrecombinant DNA molecules to direct expression of a polypeptide inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences that encode substantially the same or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express a given polypeptide.

[0120] As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

[0121] Moreover, the polynucleotide sequences of the present inventioncan be engineered using methods generally known in the art in order toalter polypeptide encoding sequences for a variety of reasons, includingbut not limited to, alterations which modify the cloning, processing,and/or expression of the gene product. For example, DNA shuffling byrandom fragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

[0122] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

[0123] Sequences encoding a desired polypeptide may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of a polypeptide, or a portionthereof. For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431 A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).

[0124] A newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.) or other comparable techniques available in theart. The composition of the synthetic peptides may be confirmed by aminoacid analysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

[0125] In order to express a desired polypeptide, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods which are well known to thoseskilled in the art may be used to construct expression vectorscontaining sequences encoding a polypeptide of interest and appropriatetranscriptional and translational control elements. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. Such techniques are described inSambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989)Current Protocols in Molecular Biology, John Wiley & Sons, New York.N.Y.

[0126] A variety of expression vector/host systems may be utilized tocontain and express polynucleotide sequences. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0127] The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

[0128] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

[0129] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0130] In cases where plant expression vectors are used, the expressionof sequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

[0131] An insect system may also be used to express a polypeptide ofinterest. For example, in one such system, Autographa califormicanuclear polyhedrosis virus (AcNPV) is used as a vector to expressforeign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.The sequences encoding the polypeptide may be cloned into anon-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe polypeptide-encoding sequence will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which the polypeptide ofinterest may be expressed (Engelhard, E. K. et al. (1994) Proc. Natl.Acad. Sci. 91 :3224-3227).

[0132] In mammalian host cells, a number of viral-based expressionsystems are generally available. For example, in cases where anadenovirus is used as an expression vector, sequences encoding apolypeptide of interest may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing the polypeptide in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0133] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding a polypeptide of interest.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

[0134] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0135] For long-term, high-yield production of recombinant proteins,stable expression is generally preferred. For example, cell lines whichstably express a polynucleotide of interest may be transformed usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

[0136] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990)Cell 22:817-23) genes which can be employed in tk.sup.- oraprt.sup.-cells, respectively. Also, antimetabolite, antibiotic orherbicide resistance can be used as the basis for selection; forexample, dhfr which confers resistance to methotrexate (Wigler, M. etal. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confersresistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin,F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which conferresistance to chlorsulfuron and phosphinotricin acetyltransferase,respectively (Murry, supra). Additional selectable genes have beendescribed, for example, trpB, which allows cells to utilize indole inplace of tryptophan, or hisD, which allows cells to utilize histinol inplace of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. 85:8047-51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins, beta-glucuronidase and itssubstrate GUS, and luciferase and its substrate luciferin, being widelyused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.55:121-131).

[0137] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0138] Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

[0139] A variety of protocols for detecting and measuring the expressionof polynucleotide-encoded products, using either polyclonal ormonoclonal antibodies specific for the product are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide may bepreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton, R. et al. (1990; Serological Methods, a LaboratoryManual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J.Exp. Med. 158:1211-1216).

[0140] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences, or any portionsthereof may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0141] Host cells transformed with a polynucleotide sequence of interestmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides ofthe invention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

[0142] In addition to recombinant production methods, polypeptides ofthe invention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

[0143] Site-specific Mutagenesis

[0144] Site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent polypeptides, through specific mutagenesis of the underlyingpolynucleotides that encode them. The technique, well-known to those ofskill in the art, further provides a ready ability to prepare and testsequence variants, for example, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site-specific mutagenesis allows the production ofmutants through the use of specific oligonucleotide sequences whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the deletion junction being traversed. Mutations may beemployed in a selected polynucleotide sequence to improve, alter,decrease, modify, or otherwise change the properties of thepolynucleotide itself, and/or alter the properties, activity,composition, stability, or primary sequence of the encoded polypeptide.

[0145] In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theantigenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

[0146] As will be appreciated by those of skill in the art,site-specific mutagenesis techniques have often employed a phage vectorthat exists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage are readily commercially-available and their useis generally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

[0147] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0148] The preparation of sequence variants of the selectedpeptide-encoding DNA segments using site-directed mutagenesis provides ameans of producing potentially useful species and is not meant to belimiting as there are other ways in which sequence variants of peptidesand the DNA sequences encoding them may be obtained. For example,recombinant vectors encoding the desired peptide sequence may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants. Specific details regarding these methods and protocols arefound in the teachings of Maloy et al., 1994; Segal, 1976; Prokop andBajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each incorporatedherein by reference, for that purpose.

[0149] As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

[0150] Polynucleotide Amplification Techniques

[0151] A number of template dependent processes are available to amplifythe target sequences of interest present in a sample. One of the bestknown amplification methods is the polymerase chain reaction (PCRTM)which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCRTM, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCRTM amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

[0152] Another method for amplification is the ligase chain reaction(referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308(specifically incorporated herein by reference in its entirety). In LCR,two complementary probe pairs are prepared, and in the presence of thetarget sequence, each pair will bind to opposite complementary strandsof the target such that they abut. In the presence of a ligase, the twoprobe pairs will link to form a single unit. By temperature cycling, asin PCR™, bound ligated units dissociate from the target and then serveas “target sequences” for ligation of excess probe pairs. U.S. Pat. No.4,883,750, incorporated herein by reference in its entirety, describesan alternative method of amplification similar to LCR for binding probepairs to a target sequence.

[0153] Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880, incorporated herein by reference in its entirety, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

[0154] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[α-thio]triphosphates in onestrand of a restriction site (Walker et al., 1992, incorporated hereinby reference in its entirety), may also be useful in the amplificationof nucleic acids in the present invention.

[0155] Strand Displacement Amplification (SDA) is another method ofcarrying out isothermal amplification of nucleic acids which involvesmultiple rounds of strand displacement and synthesis, i.e. nicktranslation. A similar method, called Repair Chain Reaction (RCR) isanother method of amplification which may be useful in the presentinvention and is involves annealing several probes throughout a regiontargeted for amplification, followed by a repair reaction in which onlytwo of the four bases are present. The other two bases can be added asbiotinylated derivatives for easy detection. A similar approach is usedin SDA.

[0156] Sequences can also be detected using a cyclic probe reaction(CPR). In CPR, a probe having a 3′ and 5′ sequences of non-target DNAand an internal or “middle” sequence of the target protein specific RNAis hybridized to DNA which is present in a sample. Upon hybridization,the reaction is treated with RNaseH, and the products of the probe areidentified as distinctive products by generating a signal that isreleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated. Thus, CPR involvesamplifying a signal generated by hybridization of a probe to a targetgene specific expressed nucleic acid.

[0157] Still other amplification methods described in Great Britain Pat.Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025, each of which is incorporated herein by reference in itsentirety, may be used in accordance with the present invention. In theformer application, “modified” primers are used in a PCR-like, templateand enzyme dependent synthesis. The primers may be modified by labelingwith a capture moiety (e.g., biotin) and/or a detector moiety (e.g.,enzyme). In the latter application, an excess of labeled probes is addedto a sample. In the presence of the target sequence, the probe binds andis cleaved catalytically. After cleavage, the target sequence isreleased intact to be bound by excess probe. Cleavage of the labeledprobe signals the presence of the target sequence.

[0158] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (Kwoh et al., 1989; PCTIntl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by referencein its entirety), including nucleic acid sequence based amplification(NASBA) and 3SR. In NASBA, the nucleic acids can be prepared foramplification by standard phenol/chloroform extraction, heatdenaturation of a sample, treatment with lysis buffer and minispincolumns for isolation of DNA and RNA or guanidinium chloride extractionof RNA. These amplification techniques involve annealing a primer thathas sequences specific to the target sequence. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat-denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target-specificprimer, followed by polymerization. The double stranded DNA moleculesare then multiply transcribed by a polymerase such as T7 or SP6. In anisothermal cyclic reaction, the RNAs are reverse transcribed into DNA,and transcribed once again with a polymerase such as T7 or SP6. Theresulting products, whether truncated or complete, indicatetarget-specific sequences.

[0159] Eur. Pat. Appl. Publ. No. 329,822, incorporated herein byreference in its entirety, disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from resultingDNA:RNA duplex by the action of ribonuclease H (RNase H, an RNasespecific for RNA in a duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

[0160] PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated hereinby reference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” (Frohman, 1990),and “one-sided PCR” (Ohara, 1989) which are well-known to those of skillin the art.

[0161] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu andDean, 1996, incorporated herein by reference in its entirety), may alsobe used in the amplification of DNA sequences of the present invention.

[0162] Biological Functional Equivalents

[0163] Modification and changes may be made in the structure of thepolynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a polypeptide with desirablecharacteristics. As mentioned above, it is often desirable to introduceone or more mutations into a specific polynucleotide sequence. Incertain circumstances, the resulting encoded polypeptide sequence isaltered by this mutation, or in other cases, the sequence of thepolypeptide is unchanged by one or more mutations in the encodingpolynucleotide.

[0164] When it is desirable to alter the amino acid sequence of apolypeptide to create an equivalent, or even an improved,second-generation molecule, the amino acid changes may be achieved bychanging one or more of the codons of the encoding DNA sequence,according to Table 1.

[0165] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. It is thus contemplated by theinventors that various changes may be made in the peptide sequences ofthe disclosed compositions, or corresponding DNA sequences which encodesaid peptides without appreciable loss of their biological utility oractivity. TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0166] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

[0167] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e. still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 (specifically incorporated herein by reference in itsentirety), states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein.

[0168] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4). It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologicallyequivalent protein. In such changes, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0169] As outlined above, amino acid substitutions are generallytherefore based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

[0170] In addition, any polynucleotide may be further modified toincrease stability in vivo. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ ends;the use of phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl-methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

[0171] In Vivo Polynucleotide Delivery Techniques

[0172] In additional embodiments, genetic constructs comprising one ormore of the polynucleotides of the invention are introduced into cellsin vivo. This may be achieved using any of a variety or well knownapproaches, several of which are outlined below for the purpose ofillustration.

[0173] 1. Adenovirus

[0174] One of the preferred methods for in vivo delivery of one or morenucleic acid sequences involves the use of an adenovirus expressionvector. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to express a polynucleotide that hasbeen cloned therein in a sense or antisense orientation. Of course, inthe context of an antisense construct, expression does not require thatthe gene product be synthesized.

[0175] The expression vector comprises a genetically engineered form ofan adenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

[0176] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target-cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP, (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0177] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0178] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses El proteins (Graham etal., 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, 1978), the current adenovirus vectors, with thehelp of 293 cells, carry foreign DNA in either the El, the D3 or bothregions (Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the El and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe El-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

[0179] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g, Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

[0180] Recently, Racher et al. (1995) disclosed improved methods forculturing 293 cells and propagating adenovirus. In one format, naturalcell aggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

[0181] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

[0182] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

[0183] Adenovirus is easy to grow and manipulate and exhibits broad hostrange in vitro and in vivo. This group of viruses can be obtained inhigh titers, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they arehighly infective. The life cycle of adenovirus does not requireintegration into the host cell genome. The foreign genes delivered byadenovirus vectors are episomal and, therefore, have low genotoxicity tohost cells. No side effects have been reported in studies of vaccinationwith wild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

[0184] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animalstudies suggested that recombinant adenovirus could be used for genetherapy (Stratford-Perricaudet and Perricaudet, 1991;Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al., 1993), peripheral intravenous injections(Herz and Gerard, 1993) and stereotactic inoculation into the brain (LeGal La Salle et al., 1993).

[0185] 2. Retroviruses

[0186] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

[0187] In order to construct a retroviral vector, a nucleic acidencoding one or more oligonucleotide or polynucleotide sequences ofinterest is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and packaging components is constructed (Mannet al., 1983). When a recombinant plasmid containing a cDNA, togetherwith the retroviral LTR and packaging sequences is introduced into thiscell line (by calcium phosphate precipitation for example), thepackaging sequence allows the RNA transcript of the recombinant plasmidto be packaged into viral particles, which are then secreted into theculture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al.,1983). The media containing the recombinant retroviruses is thencollected, optionally concentrated, and used for gene transfer.Retroviral vectors are able to infect a broad variety of cell types.However, integration and stable expression require the division of hostcells (Paskind et al., 1975).

[0188] A novel approach designed to allow specific targeting ofretrovirus vectors was recently developed based on the chemicalmodification of a retrovirus by the chemical addition of lactoseresidues to the viral envelope. This modification could permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

[0189] A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

[0190] 3. Adeno-Associated Viruses

[0191] AAV (Ridgeway, 1988; Hermonat and Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka andMcLaughlin, 1988).

[0192] The AAV DNA is approximately 4.7 kilobases long. It contains twoopen reading frames and is flanked by two ITRs (FIG. 2). There are twomajor genes in the AAV genome: rep and cap. The rep gene codes forproteins responsible for viral replications, whereas cap codes forcapsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. Theseterminal repeats are the only essential cis components of the AAV forchromosomal integration. Therefore, the AAV can be used as a vector withall viral coding sequences removed and replaced by the cassette of genesfor delivery. Three viral promoters have been identified and named p5,p19, and p40, according to their map position. Transcription from p5 andp19 results in production of rep proteins, and transcription from p40produces the capsid proteins (Hermonat and Muzyczka, 1984).

[0193] There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention.

[0194] AAV is also a good choice of delivery vehicles due to its safety.There is a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilize rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimizes immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

[0195] 4. Other Viral Vectors as Expression Constructs

[0196] Other viral vectors may be employed as expression constructs inthe present invention for the delivery of oligonucleotide orpolynucleotide sequences to a host cell. Vectors derived from virusessuch as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988),lentiviruses, polio viruses and herpes viruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al.,1990).

[0197] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

[0198] 5. Non-viral Vectors

[0199] In order to effect expression of the oligonucleotide orpolynucleotide sequences of the present invention, the expressionconstruct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. As described above, one preferred mechanism for deliveryis via viral infection where the expression construct is encapsulated inan infectious viral particle.

[0200] Once the expression construct has been delivered into the cellthe nucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

[0201] In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

[0202] Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

[0203] Selected organs including the liver, skin, and muscle tissue ofrats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin etal., 1991). This may require surgical exposure of the tissue or cells,to eliminate any intervening tissue between the gun and the targetorgan, i.e. ex vivo treatment. Again, DNA encoding a particular gene maybe delivered via this method and still be incorporated by the presentinvention.

[0204] Antisense Oligonucleotides

[0205] The end result of the flow of genetic information is thesynthesis of protein. DNA is transcribed by polymerases into messengerRNA and translated on the ribosome to yield a folded, functionalprotein. Thus there are several steps along the route where proteinsynthesis can be inhibited. The native DNA segment coding for apolypeptide described herein, as all such mammalian DNA strands, has twostrands: a sense strand and an antisense strand held together byhydrogen bonding. The messenger RNA coding for polypeptide has the samenucleotide sequence as the sense DNA strand except that the DNAthymidine is replaced by uridine. Thus, synthetic antisense nucleotidesequences will bind to a mRNA and inhibit expression of the proteinencoded by that mRNA.

[0206] The targeting of antisense oligonucleotides to mRNA is thus onemechanism to shut down protein synthesis, and, consequently, representsa powerful and targeted therapeutic approach. For example, the synthesisof polygalactauronase and the muscarine type 2 acetylcholine receptorare inhibited by antisense oligonucleotides directed to their respectivemRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829,each specifically incorporated herein by reference in its entirety).Further, examples of antisense inhibition have been demonstrated withthe nuclear protein cyclin, the multiple drug resistance gene (MDG1),ICAM-1, E-selectin, STK-1, striatal GABA_(A) receptor and human EGF(Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al.,1998; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No.5,718,709 and U.S. Pat. No. 5,610,288, each specifically incorporatedherein by reference in its entirety). Antisense constructs have alsobeen described that inhibit and can be used to treat a variety ofabnormal cellular proliferations, e.g. cancer (U.S. Pat. No. 5,747,470;U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683, each specificallyincorporated herein by reference in its entirety).

[0207] Therefore, in exemplary embodiments, the invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein.

[0208] Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence (i.e. inthese illustrative examples the rat and human sequences) anddetermination of secondary structure, Tm, binding energy, relativestabilitv, and antisense compositions were selected based upon theirrelative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell.

[0209] Highly preferred target regions of the mRNA, are those which areat or near the AUG translation initiation codon, and those sequenceswhich were substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationswere performed using v.4 of the OLIGO primer analysis software (Rychlik,1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

[0210] The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp4l and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., 1997). It has been demonstrated thatseveral molecules of the MPG peptide coat the antisense oligonucleotidesand can be delivered into cultured mammalian cells in less than 1 hourwith relatively high efficiency (90%). Further, the interaction with MPGstrongly increases both the stability of the oligonucleotide to nucleaseand the ability to cross the plasma membrane (Morris et al., 1997).

[0211] Ribozymes

[0212] Although proteins traditionally have been used for catalysis ofnucleic acids, another class of macromolecules has emerged as useful inthis endeavor. Ribozymes are RNA-protein complexes that cleave nucleicacids in a site-specific fashion. Ribozymes have specific catalyticdomains that possess endonuclease activity (Kim and Cech, 1987; Gerlachet al., 1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

[0213] Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein by reference) reports that certainribozymes can act as endonucleases with a sequence specificity greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

[0214] Six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

[0215] The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

[0216] The enzymatic nucleic acid molecule may be formed in ahammerhead, hairpin, a hepatitis δ virus, group I intron or RNaseP RNA(in association with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992).Examples of hairpin motifs are described by Hampel etal. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel etal.(1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein byreference). An example of the hepatitis δ virus motif is described byPerrotta and Been (1992); an example of the RNaseP motif is described byGuerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif isdescribed by Collins (Saville and Collins, 1990; Saville and Collins,1991; Collins and Olive, 1993); and an example of the Group I intron isdescribed in (U.S. Pat. No. 4,987,071, specifically incorporated hereinby reference). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

[0217] In certain embodiments, it may be important to produce enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required.Alternatively, the ribozymes can be expressed from DNA or RNA vectorsthat are delivered to specific cells.

[0218] Small enzymatic nucleic acid motifs (e.g., of the hammerhead orthe hairpin structure) may also be used for exogenous delivery. Thesimple structure of these molecules increases the ability of theenzymatic nucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl.Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa etal., 1992; Taira et al., 1991; and Ventura et al., 1993).

[0219] Ribozymes may be added directly, or can be complexed withcationic lipids, lipid complexes, packaged within liposomes, orotherwise delivered to target cells. The RNA or RNA complexes can belocally administered to relevant tissues ex vivo, or in vivo throughinjection, aerosol inhalation, infusion pump or stent, with or withouttheir incorporation in biopolymers.

[0220] Ribozymes may be designed as described in Int. Pat. Appl. Publ.No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, eachspecifically incorporated herein by reference) and synthesized to betested in vitro and in vivo, as described. Such ribozymes can also beoptimized for delivery. While specific examples are provided, those inthe art will recognize that equivalent RNA targets in other species canbe utilized when necessary.

[0221] Hammerhead or hairpin ribozymes may be individually analyzed bycomputer folding (Jaeger et al., 1989) to assess whether the ribozymesequences fold into the appropriate secondary structure. Those ribozymeswith unfavorable intramolecular interactions between the binding armsand the catalytic core are eliminated from consideration. Varyingbinding arm lengths can be chosen to optimize activity. Generally, atleast 5 or so bases on each arm are able to bind to, or otherwiseinteract with, the target RNA.

[0222] Ribozymes of the hammerhead or hairpin motif may be designed toanneal to various sites in the mRNA message, and can be chemicallysynthesized. The method of synthesis used follows the procedure fornormal RNA synthesis as described in Usman etal. (1987) and in Scaringeetal. (1990) and makes use of common nucleic acid protecting andcoupling groups, such as dimethoxytrityl at the 5′-end, andphosphoramidites at the 3′-end. Average stepwise coupling yields aretypically >98%. Hairpin ribozymes may be synthesized in two parts andannealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).Ribozymes may be modified extensively to enhance stability bymodification with nuclease resistant groups, for example, 2′-amino,2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H (for a review see e.g., Usmanand Cedergren, 1992). Ribozymes may be purified by gel electrophoresisusing general methods or by high pressure liquid chromatography andresuspended in water.

[0223] Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No.92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

[0224] Sullivan etal (Int. Pat. Appl. Publ. No. WO 94/02595) describesthe general methods for delivery of enzymatic RNA molecules. Ribozymesmay be administered to cells by a variety of methods known to thosefamiliar to the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

[0225] Another means of accumulating high concentrations of aribozyme(s) within cells is to incorporate the ribozyme-encodingsequences into a DNA expression vector. Transcription of the ribozymesequences are driven from a promoter for eukaryotic RNA polymerase I(pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III).Transcripts from pol II or pol III promoters will be expressed at highlevels in all cells; the levels of a given pol II promoter in a givencell type will depend on the nature of the gene regulatory sequences(enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerasepromoters may also be used, providing that the prokaryotic RNApolymerase enzyme is expressed in the appropriate cells (Elroy-Stein andMoss, 1990; Gao and Huang, 1993; Lieber et al., 1993; Zhou etal., 1990).Ribozymes expressed from such promoters can function in mammalian cells(e.g Kashani-Saber etal., 1992; Ojwang etal., 1992; Chen etal., 1992; Yuetal., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993). Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

[0226] Ribozymes may be used as diagnostic tools to examine geneticdrift and mutations within diseased cells. They can also be used toassess levels of the target RNA molecule. The close relationship betweenribozyme activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple ribozymes, one may map nucleotide changes which are importantto RNA structure and function in vitro, as well as in cells and tissues.Cleavage of target RNAs with ribozymes may be used to inhibit geneexpression and define the role (essentially) of specified gene productsin the progression of disease. In this manner, other genetic targets maybe defined as important mediators of the disease. These studies willlead to better treatment of the disease progression by affording thepossibility of combinational therapies (e.g., multiple ribozymestargeted to different genes, ribozymes coupled with known small moleculeinhibitors, or intermittent treatment with combinations of ribozymesand/or other chemical or biological molecules). Other in vitro uses ofribozymes are well known in the art, and include detection of thepresence of mRNA associated with an IL-5 related condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

[0227] Peptide Nucleic Acids

[0228] In certain embodiments, the inventors contemplate the use ofpeptide nucleic acids (PNAs) in the practice of the methods of theinvention. PNA is a DNA mimic in which the nucleobases are attached to apseudopeptide backbone (Good and Nielsen, 1997). PNA is able to beutilized in a number methods that traditionally have used RNA or DNA.Often PNA sequences perform better in techniques than the correspondingRNA or DNA sequences and have utilities that are not inherent to RNA orDNA. A review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (1997) and is incorporatedherein by reference. As such, in certain embodiments, one may preparePNA sequences that are complementary to one or more portions of the ACEmRNA sequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

[0229] PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., 1991; Hanvey et al.,1992; Hyrup and Nielsen, 1996; Neilsen, 1996). This chemistry has threeimportant consequences: firstly, in contrast to DNA or phosphorothioateoligonucleotides, PNAs are neutral molecules; secondly, PNAs areachiral, which avoids the need to develop a stereoselective synthesis;and thirdly, PNA synthesis uses standard Boc (Dueholm et al., 1994) orFmoc (Thomson et al., 1995) protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used (Christensen et al., 1995).

[0230] PNA monomers or ready-made oligomers are commercially availablefrom PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., 1995). The manual protocol lends itself to theproduction of chemically modified PNAs or the simultaneous synthesis offamilies of closely related PNAs.

[0231] As with peptide synthesis, the success of a particular PNAsynthesis will depend on the properties of the chosen sequence. Forexample, while in theory PNAs can incorporate any combination ofnucleotide bases, the presence of adjacent purines can lead to deletionsof one or more residues in the product. In expectation of thisdifficulty, it is suggested that, in producing PNAs with adjacentpurines, one should repeat the coupling of residues likely to be addedinefficiently. This should be followed by the purification of PNAs byreverse-phase high-pressure liquid chromatography (Norton et al., 1995)providing yields and purity of product similar to those observed duringthe synthesis of peptides.

[0232] Modifications of PNAs for a given application may be accomplishedby coupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (Norton et al., 1995;Haaima et al., 1996; Stetsenko et al., 1996; Petersen et al., 1995;Ulmann et al., 1996; Koch et al., 1995; Orum et al., 1995; Footer etal., 1996; Griffith et al., 1995; Kremsky et al., 1996; Pardridge etal., 1995; Boffa et al., 1995; Landsdorp et al., 1996;Gambacorti-Passerini et al., 1996; Armitage et al., 1997; Seeger et al.,1997; Ruskowski et al., 1997). U.S. Pat. No. 5,700,922 discussesPNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulatingprotein in organisms, and treatment of conditions susceptible totherapeutics.

[0233] In contrast to DNA and RNA, which contain negatively chargedlinkages, the PNA backbone is neutral. In spite of this dramaticalteration, PNAs recognize complementary DNA and RNA by Watson-Crickpairing (Egholm et al., 1993), validating the initial modeling byNielsen et al. (1991). PNAs lack 3′ to 5′ polarity and can bind ineither parallel or antiparallel fashion, with the antiparallel modebeing preferred (Egholm et al., 1993).

[0234] Hybridization of DNA oligonucleotides to DNA and RNA isdestabilized by electrostatic repulsion between the negatively chargedphosphate backbones of the complementary strands. By contrast, theabsence of charge repulsion in PNA-DNA or PNA-RNA duplexes increases themelting temperature (Tm) and reduces the dependence of Tm on theconcentration of mono- or divalent cations (Nielsen et al., 1991). Theenhanced rate and affinity of hybridization are significant because theyare responsible for the surprising ability of PNAs to perform strandinvasion of complementary sequences within relaxed double-stranded DNA.In addition, the efficient hybridization at inverted repeats suggeststhat PNAs can recognize secondary structure effectively withindouble-stranded DNA. Enhanced recognition also occurs with PNAsimmobilized on surfaces, and Wang et al. have shown that support-boundPNAs can be used to detect hybridization events (Wang et al., 1996).

[0235] One might expect that tight binding of PNAs to complementarysequences would also increase binding to similar (but not identical)sequences, reducing the sequence specificity of PNA recognition. As withDNA hybridization, however, selective recognition can be achieved bybalancing oligomer length and incubation temperature. Moreover,selective hybridization of PNAs is encouraged by PNA-DNA hybridizationbeing less tolerant of base mismatches than DNA-DNA hybridization. Forexample, a single mismatch within a 16 bp PNA-DNA duplex can reduce theTm by up to 15° C. (Egholm et al., 1993). This high level ofdiscrimination has allowed the development of several PNA-basedstrategies for the analysis of point mutations (Wang et al., 1996;Carlsson et al., 1996; Thiede et al., 1996; Webb and Hurskainen, 1996;Perry-O'Keefe et al., 1996).

[0236] High-affinity binding provides clear advantages for molecularrecognition and the development of new applications for PNAs. Forexample, 11-13 nucleotide PNAs inhibit the activity of telomerase, aribonucleo-protein that extends telomere ends using an essential RNAtemplate, while the analogous DNA oligomers do not (Norton et al.,1996).

[0237] Neutral PNAs are more hydrophobic than analogous DNA oligomers,and this can lead to difficulty solubilizing them at neutral pH,especially if the PNAs have a high purine content or if they have thepotential to form secondary structures. Their solubility can be enhancedby attaching one or more positive charges to the PNA termini (Nielsen etal., 1991).

[0238] Findings by Allfrey and colleagues suggest that strand invasionwill occur spontaneously at sequences within chromosomal DNA (Boffa etal., 1995; Boffa et al., 1996). These studies targeted PNAs to tripletrepeats of the nucleotides CAG and used this recognition to purifytranscriptionally active DNA (Boffa et al., 1995) and to inhibittranscription (Boffa et al., 1996). This result suggests that if PNAscan be delivered within cells then they will have the potential to begeneral sequence-specific regulators of gene expression. Studies andreviews concerning the use of PNAs as antisense and anti-gene agentsinclude Nielsen et al. (1993b), Hanvey et al. (1992), and Good andNielsen (1997). Koppelhus et al. (1997) have used PNAs to inhibit HIV-1inverse transcription, showing that PNAs may be used for antiviraltherapies.

[0239] Methods of characterizing the antisense binding properties ofPNAs are discussed in Rose (1993) and Jensen et aL (1997). Rose usescapillary gel electrophoresis to determine binding of PNAs to theircomplementary oligonucleotide, measuring the relative binding kineticsand stoichiometry. Similar types of measurements were made by Jensen etal. using BIAcore™ technology.

[0240] Other applications of PNAs include use in DNA strand invasion(Nielsen et al., 1991), antisense inhibition (Hanvey et al., 1992),mutational analysis (Orum et al., 1993), enhancers of transcription(Mollegaard et al., 1994), nucleic acid purification (Orum et al.,1995), isolation of transcriptionally active genes (Boffa et al., 1995),blocking of transcription factor binding (Vickers et al., 1995), genomecleavage (Veselkov et al., 1996), biosensors (Wang et al., 1996), insitu hybridization (Thisted et al., 1996), and in a 5 alternative toSouthern blotting (Perry-O'Keefe, 1996).

[0241] Polypeptide Compositions

[0242] The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from HSV. Preferably, the polypeptide is encoded by apolynucleotide sequence disclosed herein or a sequence which hybridizesunder moderate or highly stringent conditions to a polynucleotidesequence disclosed herein. Alternatively, the polypeptide may be definedas a polypeptide which comprises a contiguous amino acid sequence froman amino acid sequence disclosed herein, or which polypeptide comprisesan entire amino acid sequence disclosed herein.

[0243] In the present invention, a polypeptide composition is alsounderstood to comprise one or more polypeptides that are immunologicallyreactive with antibodies and/or T cells generated against a polypeptideof the invention, particularly a polypeptide having amino acid sequencesdisclosed herein, or to active fragments, or to variants or biologicalfunctional equivalents thereof.

[0244] Likewise, a polypeptide composition of the present invention isunderstood to comprise one or more polypeptides that are capable ofeliciting antibodies or T cells that are immunologically reactive withone or more polypeptides encoded by one or more contiguous nucleic acidsequences contained in the amino acid sequences disclosed herein, or toactive fragments, or to variants thereof, or to one or more nucleic acidsequences which hybridize to one or more of these sequences underconditions of moderate to high stringency. Particularly illustrativepolypeptides comprise the amino acid sequence disclosed in SEQ ID NO: 2,5, 6, 9, 10, 11, 14, 17, 20, 21, 22 and 25.

[0245] As used herein, an active fragment of a polypeptide includes awhole or a portion of a polypeptide which is modified by conventionaltechniques, e.g., mutagenesis, or by addition, deletion, orsubstitution, but which active fragment exhibits substantially the samestructure function, antigenicity, etc., as a polypeptide as describedherein.

[0246] In certain illustrative embodiments, the polypeptides of theinvention will comprise at least an immunogenic portion of an HSVantigen or a variant or biological functional equivalent thereof, asdescribed herein. Polypeptides as described herein may be of any length.Additional sequences derived from the native protein and/or heterologoussequences may be present, and such sequences may (but need not) possessfurther immunogenic or antigenic properties.

[0247] An “immunogenic portion,” as used herein is a portion of aprotein that is recognized (i.e., specifically bound) by a B-cell and/orT-cell surface antigen receptor. Such immunogenic portions generallycomprise at least 5 amino acid residues, more preferably at least 10,and still more preferably at least 20 amino acid residues of an HSVprotein or a variant thereof. Certain preferred immunogenic portionsinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other preferred immunogenicportions may contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

[0248] Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of anative HSV protein is a portion that reacts with such antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Such immunogenic portions may react within such assays at alevel that is similar to or greater than the reactivity of the fulllength polypeptide. Such screens may generally be performed usingmethods well known to those of ordinary skill in the art, such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. For example, a polypeptide may beimmobilized on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilized polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A.

[0249] As noted above, a composition may comprise a variant of a nativeHSV protein. A polypeptide “variant,” as used herein, is a polypeptidethat differs from a native HSV protein in one or more substitutions,deletions, additions and/or insertions, such that the immunogenicity ofthe polypeptide is not substantially diminished. In other words, theability of a variant to react with antigen-specific antisera may beenhanced or unchanged, relative to the native protein, or may bediminished by less than 50%, and preferably less than 20%, relative tothe native protein. Such variants may generally be identified bymodifying one of the above polypeptide sequences and evaluating thereactivity of the modified polypeptide with antigen-specific antibodiesor antisera as described herein. Preferred variants include those inwhich one or more portions, such as an N-terminal leader sequence ortransmembrane domain, have been removed. Other preferred variantsinclude variants in which a small portion (e.g., 1-30 amino acids,preferably 5-15 amino acids) has been removed from the N- and/orC-terminal of the mature protein.

[0250] Polypeptide variants encompassed by the present invention includethose exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined asdescribed above) to the polypeptides disclosed herein.

[0251] Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

[0252] As noted above, polypeptides may comprise a signal (or leader)sequence at the N-terminal end of the protein, which co-translationallyor post-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

[0253] Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

[0254] Portions and other variants having less than about 100 aminoacids, and generally less than about 50 amino acids, may also begenerated by synthetic means, using techniques well known to those ofordinary skill in the art. For example, such polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

[0255] Within certain specific embodiments, a polypeptide may be afusion protein that comprises multiple polypeptides as described herein,or that comprises at least one polypeptide as described herein and anunrelated sequence, such as a known protein. A fusion partner may, forexample, assist in providing T helper epitopes (an immunological fusionpartner), preferably T helper epitopes recognized by humans, or mayassist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

[0256] Fusion proteins may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusion proteinis expressed as a recombinant protein, allowing the production ofincreased levels, relative to a non-fused protein, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion protein that retains the biological activity ofboth component polypeptides.

[0257] A peptide linker sequence may be employed to separate the firstand second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

[0258] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

[0259] Fusion proteins are also provided. Such proteins comprise apolypeptide as described herein together with an unrelated immunogenicprotein. Preferably the immunogenic protein is capable of eliciting arecall response. Examples of such proteins include tetanus, tuberculosisand hepatitis proteins (see, for example, Stoute et al. New Engl. J.Med., 336:86-91, 1997).

[0260] Within preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

[0261] In another embodiment, a Mycobacterium tuberculosis-derived Ra12polynucleotide is linked to at least an immunogenic portion of an HSVpolynucleotide of this invention. Ra12 compositions and methods fortheir use in enhancing expression of heterologous polynucleotidesequences is described in U.S. Patent Application No. 60/158,585, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, Ra12 refers to a polynucleotide region that is a subsequence ofa Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serineprotease of 32 KD molecular weight encoded by a gene in virulent andavirulent strains of M tuberculosis. The nucleotide sequence and aminoacid sequence of MTB32A have been disclosed (U.S. Patent Application No.60/158,585; see also, Skeiky et al., Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). The Ra12 C-terminalfragment of the MTB32A coding sequence expresses at high levels on itsown and remains as a soluble protein throughout the purificationprocess. Moreover, the presence of Ra12 polypeptide fragments in afusion polypeptide may enhance the immunogenicity of the heterologousantigenic HSV polypeptides with which Ra12 is fused. In one embodiment,the Ra12 polypeptide sequence present in a fusion polypeptide with anHSV antigen comprises some or all of amino acid residues 192 to 323 ofMTB32A.

[0262] In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably a C-terninalportion). LYTA is derived from Streptococcus pneumoniae, whichsynthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encodedby the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin thatspecifically degrades certain bonds in the peptidoglycan backbone. TheC-terminal domain of the LYTA protein is responsible for the affinity tothe choline or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

[0263] In general, polypeptides (including fusion proteins) andpolynucleotides as described herein are isolated. An “isolated”polypeptide or polynucleotide is one that is removed from its originalenvironment. For example, a naturally-occurring protein is isolated ifit is separated from some or all of the coexisting materials in thenatural system. Preferably, such polypeptides are at least about 90%pure, more preferably at least about 95% pure and most preferably atleast about 99% pure. A polynucleotide is considered to be isolated if,for example, it is cloned into a vector that is not a part of thenatural environment.

[0264] Binding Agents

[0265] The present invention further provides agents, such as antibodiesand antigen-binding fragments thereof, that specifically bind to a HSVprotein. As used herein, an antibody, or antigen-binding fragmentthereof, is said to “specifically bind” to a HSV protein if it reacts ata detectable level (within, for example, an ELISA) with a HSV protein,and does not react detectably with unrelated proteins under similarconditions. As used herein, “binding” refers to a noncovalentassociation between two separate molecules such that a complex isformed. The ability to bind may be evaluated by, for example,determining a binding constant for the formation of the complex. Thebinding constant is the value obtained when the concentration of thecomplex is divided by the product of the component concentrations. Ingeneral, two compounds are said to “bind,” in the context of the presentinvention, when the binding constant for complex formation exceeds about10³ L/mol. The binding constant may be determined using methods wellknown in the art.

[0266] Binding agents may be further capable of differentiating betweenpatients with and without HSV infection using the representative assaysprovided herein. For example, preferably, antibodies or other bindingagents that bind to a HSV protein will generate a signal indicating thepresence of infection in at least about 20% of patients with thedisease, and will generate a negative signal indicating the absence ofthe disease in at least about 90% of individuals without an HSVinfection. To determine whether a binding agent satisfies thisrequirement, biological samples (e.g., blood, sera, sputum, urine and/orbiopsies) from patients with and without HSV (as determined usingstandard clinical tests) may be assayed as described herein for thepresence of polypeptides that bind to the binding agent. It will beapparent that a statistically significant number of samples with andwithout the disease should be assayed. Each binding agent should satisfythe above criteria; however, those of ordinary skill in the art willrecognize that binding agents may be used in combination to improvesensitivity.

[0267] Any agent that satisfies the above requirements may be a bindingagent. For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

[0268] Monoclonal antibodies specific for an antigenic polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

[0269] Monoclonal antibodies may be isolated from the supernatants ofgrowing hybridoma colonies. In addition, various techniques may beemployed to enhance the yield, such as injection of the hybridoma cellline into the peritoneal cavity of a suitable vertebrate host, such as amouse. Monoclonal antibodies may then be harvested from the ascitesfluid or the blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

[0270] Within certain embodiments, the use of antigen-binding fragmentsof antibodies may be preferred. Such fragments include Fab fragments,which may be prepared using standard techniques. Briefly,immunoglobulins may be purified from rabbit serum by affinitychromatography on Protein A bead columns (Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns.

[0271] Monoclonal antibodies of the present invention may be coupled toone or more therapeutic agents. Suitable agents in this regard includeradionuclides, differentiation inducers, drugs, toxins, and derivativesthereof. Preferred radionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi. Preferred drugs include methotrexate, andpyrimidine and purine analogs. Preferred differentiation inducersinclude phorbol esters and butyric acid. Preferred toxins include ricin,abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin,Shigella toxin, and pokeweed antiviral protein.

[0272] A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

[0273] Alternatively, it may be desirable to couple a therapeutic agentand an antibody via a linker group. A linker group can function as aspacer to distance an antibody from an agent in order to avoidinterference with binding capabilities. A linker group can also serve toincrease the chemical reactivity of a substituent on an agent or anantibody, and thus increase the coupling efficiency. An increase inchemical reactivity may also facilitate the use of agents, or functionalgroups on agents, which otherwise would not be possible.

[0274] It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

[0275] Where a therapeutic agent is more potent when free from theantibody portion of the immunoconjugates of the present invention, itmay be desirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

[0276] It may be desirable to couple more than one agent to an antibody.In one embodiment, multiple molecules of an agent are coupled to oneantibody molecule. In another embodiment, more than one type of agentmay be coupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

[0277] A carrier may bear the agents in a variety of ways, includingcovalent bonding either directly or via a linker group. Suitablecarriers include proteins such as albumins (e.g., U.S. Pat. No.4,507,234, to Kato et al.), peptides and polysaccharides such asaminodextran (e.g., U.S. Pat. No. 4,699,784, to Shih et al.). A carriermay also bear an agent by noncovalent bonding or by encapsulation, suchas within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and4,873,088). Carriers specific for radionuclide agents includeradiohalogenated small molecules and chelating compounds. For example,U.S. Pat. No. 4,735,792 discloses representative radiohalogenated smallmolecules and their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen and sulfuratoms as the donor atoms for binding the metal, or metal oxide,radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al.discloses representative chelating compounds and their synthesis.

[0278] A variety of routes of administration for the antibodies andimmunoconjugates may be used. Typically, administration will beintravenous, intramuscular, subcutaneous and the like. It will beevident that the precise dose of the antibody/immunoconjugate will varydepending upon the antibody used, the antigen density, and the rate ofclearance of the antibody.

[0279] T Cells

[0280] Immunotherapeutic compositions may also, or alternatively,comprise T cells specific for HSV protein. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood, or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the IsolexTM System, availablefrom Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human mammals, cell lines or cultures.

[0281] T cells may be stimulated with a HSV polypeptide, polynucleotideencoding a HSV polypeptide and/or an antigen presenting cell (APC) thatexpresses such a polypeptide. Such stimulation is performed underconditions and for a time sufficient to permit the generation of T cellsthat are specific for the polypeptide. In certain embodiments, HSVpolypeptide or polynucleotide is present within a delivery vehicle, suchas a microsphere, to facilitate the generation of specific T cells.

[0282] T cells are considered to be specific for a HSV polypeptide ifthe T cells specifically proliferate, secrete cytokines or kill targetcells coated with the polypeptide or expressing a gene encoding thepolypeptide. T cell specificity may be evaluated using any of a varietyof standard techniques. For example, within a chromium release assay orproliferation assay, a stimulation index of more than two fold increasein lysis and/or proliferation, compared to negative controls, indicatesT cell specificity. Such assays may be performed, for example, asdescribed in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively,detection of the proliferation of T cells may be accomplished by avariety of known techniques. For example, T cell proliferation can bedetected by measuring an increased rate of DNA synthesis (e.g., bypulse-labeling cultures of T cells with tritiated thymidine andmeasuring the amount of tritiated thymidine incorporated into DNA).Contact with a HSV polypeptide (100 ng/ml-100 μg/ml, preferably 200ng/ml -25 μg/ml) for 3-7 days should result in at least a two foldincrease in proliferation of the T cells. Contact as described above for2-3 hours should result in activation of the T cells, as measured usingstandard cytokine assays in which a two fold increase in the level ofcytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation(see Coligan et al., Current Protocols in Immunology, vol. 1, WileyInterscience (Greene 1998)). T cells that have been activated inresponse to a HSV polypeptide, polynucleotide or polypeptide-expressingAPC may be CD4⁺ and/or CD8⁺. HSV protein-specific T cells may beexpanded using standard techniques. Within preferred embodiments, the Tcells are derived from a patient, a related donor or an unrelated donor,and are administered to the patient following stimulation and expansion.

[0283] For therapeutic purposes, CD4⁺ or CD8⁺T cells that proliferate inresponse to a HSV polypeptide, polynucleotide or APC can be expanded innumber either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a HSV polypeptide, or a short peptide correspondingto an immunogenic portion of such a polypeptide, with or without theaddition of T cell growth factors, such as interleukin-2, and/orstimulator cells that synthesize a HSV polypeptide. Alternatively, oneor more T cells that proliferate in the presence of a HSV protein can beexpanded in number by cloning. Methods for cloning cells are well knownin the art, and include limiting dilution.

[0284] Pharmaceutical Compositions

[0285] In additional embodiments, the present invention concernsformulation of one or more of the polynucleotide, polypeptide, T-celland/or antibody compositions disclosed herein inpharmaceutically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy.

[0286] It will also be understood that, if desired, the nucleic acidsegment, RNA, DNA or PNA compositions that express a polypeptide asdisclosed herein may be administered in combination with other agents aswell, such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

[0287] Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

[0288] 1. Oral Delivery

[0289] In certain applications, the pharmaceutical compositionsdisclosed herein may be delivered via oral administration to an animal.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0290] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitzet al., 1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup of elixir may contain the active compound sucrose as asweetening agent methyl and propylparabens as preservatives, a dye andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

[0291] Typically, these formulations may contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0292] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation. For example, a mouthwash maybe prepared incorporating the active ingredient in the required amountin an appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

[0293] 2. Injectable Delivery

[0294] In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0295] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifingal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0296] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, a sterile aqueous medium that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, and the general safety and puritystandards as required by FDA Office of Biologics standards.

[0297] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0298] The compositions disclosed herein may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

[0299] As used herein, “carrier” includes any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0300] The phrase “pharmaceutically-acceptable” refers to molecularentities and compositions that do not produce an allergic or similaruntoward reaction when administered to a human. The preparation of anaqueous composition that contains a protein as an active ingredient iswell understood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

[0301] 3. Nasal Delivery

[0302] In certain embodiments, the pharmaceutical compositions may bedelivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No.5,804,212 (each specifically incorporated herein by reference in itsentirety). Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

[0303] 4. Liposome-, Nanocapsule-, and Microparticle-mediated Delivery

[0304] In certain embodiments, the inventors contemplate the use ofliposomes, nanocapsules, microparticles, microspheres, lipid particles,vesicles, and the like, for the introduction of the compositions of thepresent invention into suitable host cells. In particular, thecompositions of the present invention may be formulated for deliveryeither encapsulated in a lipid particle, a liposome, a vesicle, ananosphere, or a nanoparticle or the like.

[0305] Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434;U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No.5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporatedherein by reference in its entirety).

[0306] Liposomes have been used successfully with a number of cell typesthat are normally resistant to transfection by other proceduresincluding T cell suspensions, primary hepatocyte cultures and PC 12cells (Renneisen et al., 1990; Muller et al., 1990). In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs (Heath and Martin, 1986; Heath et al., 1986;Balazsovits et al., 1989; Fresta and Puglisi, 1996), radiotherapeuticagents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumiet al., 1990b), viruses (Faller and Baltimore, 1984), transcriptionfactors and allosteric effectors (Nicolau and Gersonde, 1979) into avariety of cultured cell lines and animals. In addition, severalsuccessful clinical trails examining the effectiveness ofliposome-mediated drug delivery have been completed (Lopez-Berestein etal., 1985a; 1985b; Coune, 1988; Sculier et al., 1988). Furthermore,several studies suggest that the use of liposomes is not associated withautoimmune responses, toxicity or gonadal localization after systemicdelivery (Mori and Fukatsu, 1992).

[0307] Liposomes are formed from phospholipids that are dispersed in anaqueous medium and spontaneously form multilamellar concentric bilayervesicles (also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

[0308] Liposomes bear resemblance to cellular membranes and arecontemplated for use in connection with the present invention ascarriers for the peptide compositions. They are widely suitable as bothwater- and lipid-soluble substances can be entrapped, i.e. in theaqueous spaces and within the bilayer itself, respectively. It ispossible that the drug-bearing liposomes may even be employed forsite-specific delivery of active agents by selectively modifying theliposomal formulation.

[0309] In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

[0310] In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

[0311] The ability to trap solutes varies between different types ofliposomes. For example, MLVs are moderately efficient at trappingsolutes, but SUVs are extremely inefficient. SUVs offer the advantage ofhomogeneity and reproducibility in size distribution, however, and acompromise between size and trapping efficiency is offered by largeunilamellar vesicles (LUVs). These are prepared by ether evaporation andare three to four times more efficient at solute entrapment than MLVs.

[0312] In addition to liposome characteristics, an important determinantin entrapping compounds is the physicochemical properties of thecompound itself. Polar compounds are trapped in the aqueous spaces andnonpolar compounds bind to the lipid bilayer of the vesicle. Polarcompounds are released through permeation or when the bilayer is broken,but nonpolar compounds remain affiliated with the bilayer unless it isdisrupted by temperature or exposure to lipoproteins. Both types showmaximum efflux rates at the phase transition temperature.

[0313] Liposomes interact with cells via four different mechanisms:endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and neutrophils; adsorption to the cell surface, eitherby nonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

[0314] The fate and disposition of intravenously injected liposomesdepend on their physical properties, such as size, fluidity, and surfacecharge. They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

[0315] Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

[0316] Alternatively, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (Henry-Michelland et al., 1987;Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkylcyanoacrylatenanoparticles that meet these requirements are contemplated for use inthe present invention. Such particles may be are easily made, asdescribed (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambauxet al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684,specifically incorporated herein by reference in its entirety).

[0317] Vaccines

[0318] In certain preferred embodiments of the present invention,vaccines are provided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, M. F. Powell and M.J. Newman, eds., “Vaccine Design (the subunit and adjuvant approach),”Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines withinthe scope of the present invention may also contain other compounds,which may be biologically active or inactive. For example, one or moreimmunogenic portions of other HSV antigens may be present, eitherincorporated into a fusion polypeptide or as a separate compound, withinthe composition or vaccine.

[0319] Illustrative vaccines may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. As noted above, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Numerous gene delivery techniques are well known in the art,such as those described by Rolland, Crit. Rev. Therap. Drug CarrierSystems 15:143-198, 1998, and references cited therein. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium (such as Bacillus-Calmette-Guerrin) that expresses animmunogenic portion of the polypeptide on its cell surface or secretessuch an epitope. In a preferred embodiment, the DNA may be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci.569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells. It will be apparent that a vaccine may comprise both apolynucleotide and a polypeptide component. Such vaccines may providefor an enhanced immune response.

[0320] It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g., sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

[0321] While any suitable carrier known to those of ordinary skill inthe art may be employed in the vaccine compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. Modified hepatitis B core protein carriersystems are also suitable, such as those described in WO/99 40934, andreferences cited therein, all incorporated herein by reference. One mayalso employ a carrier comprising the particulate-protein complexesdescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

[0322] Such compositions may also comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, bacteriostats, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminumhydroxide), solutes that render the formulation isotonic, hypotonic orweakly hypertonic with the blood of a recipient, suspending agents,thickening agents and/or preservatives. Alternatively, compositions ofthe present invention may be formulated as a lyophilizate. Compounds mayalso be encapsulated within liposomes using well known technology.

[0323] Any of a variety of immunostimulants may be employed in thevaccines of this invention. For example, an adjuvant may be included.Most adjuvants contain a substance designed to protect the antigen fromrapid catabolism, such as aluminum hydroxide or mineral oil, and astimulator of immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Suitable adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2,-7, or -12, may also be used as adjuvants.

[0324] Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g. IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g. IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoralimmune responses. Following application of a vaccine as provided herein,a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

[0325] Preferred adjuvants for use in eliciting a predominantly Thl-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, WA; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc.,Framingham, Mass.), which may be used alone or in combination with otheradjuvants. For example, an enhanced system involves the combination of amonophosphoryl lipid A and saponin derivative, such as the combinationof QS21 and 3D-MPL as described in WO 94/00153, or a less reactogeniccomposition where the QS21 is quenched with cholesterol, as described inWO 96/33739. Other preferred formulations comprise an oil-in-wateremulsion and tocopherol. A particularly potent adjuvant formulationinvolving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion isdescribed in WO 95/17210.

[0326] Other preferred adjuvants include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g, SBAS-2 or SBAS-4, availablefrom SmithKline Beecham, Rixensart, Belgium), Detox (Corixa, Hamilton,Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties. Other preferred adjuvants comprise polyoxyethylene ethers,such as those described in WO 99/52549A1.

[0327] Any vaccine provided herein may be prepared using well knownmethods that result in a combination of antigen, immune responseenhancer and a suitable carrier or excipient. The compositions describedherein may be administered as part of a sustained release formulation(i.e., a formulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438, 1996) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

[0328] Carriers for use within such formulations are biocompatible, andmay also be biodegradable; preferably the formulation provides arelatively constant level of active component release. Such carriersinclude microparticles of poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose, dextran and the like. Other delayed-releasecarriers include supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

[0329] Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets HSV-infected cells.Delivery vehicles include antigen presenting cells (APCs), such asdendritic cells, macrophages, B cells, monocytes and other cells thatmay be engineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-HSV effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0330] Certain preferred embodiments of the present invention usedendritic cells or progenitors thereof as antigen-presenting cells.Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticimmunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). Ingeneral, dendritic cells may be identified based on their typical shape(stellate in situ, with marked cytoplasmic processes (dendrites) visiblein vitro), their ability to take up, process and present antigens withhigh efficiency and their ability to activate naive T cell responses.Dendritic cells may, of course, be engineered to express specificcell-surface receptors or ligands that are not commonly found ondendritic cells in vivo or ex vivo, and such modified dendritic cellsare contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

[0331] Dendritic cells and progenitors may be obtained from peripheralblood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood orany other suitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

[0332] Dendritic cells are conveniently categorized as “immature” and“mature” cells, which allows a simple way to discriminate between twowell characterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcy receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

[0333] APCs may generally be transfected with a polynucleotide encodinga HSV protein (or portion or other variant thereof) such that the HSVpolypeptide, or an immunogenic portion thereof, is expressed on the cellsurface. Such transfection may take place ex vivo, and a composition orvaccine comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the HSV polypeptide, DNA (nakedor within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

[0334] Vaccines and pharmaceutical compositions may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are preferably hermetically sealed to preserve sterilityof the formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

[0335] Immunotherapeutic Applications

[0336] In further aspects of the present invention, the compositionsdescribed herein may be used for immunotherapy of HSV infections. Withinsuch methods, pharmaceutical compositions and vaccines are typicallyadministered to a patient. As used herein, a “patient” refers to anywarm-blooded animal, preferably a human. The above pharmaceuticalcompositions and vaccines may be used to prophylactically prevent orameliorate the extent of infection by HSV or to treat a patient alreadyinfected with HSV. Administration may be by any suitable method,including administration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical, and oralroutes.

[0337] Within certain embodiments, immunotherapy may be activeimmunotherapy, in which treatment relies on the in vivo stimulation ofthe endogenous host immune system to react against HSV infection withthe administration of immune response-modifying agents (such aspolypeptides and polynucleotides as provided herein).

[0338] Within other embodiments, immunotherapy may be passiveimmunotherapy, in which treatment involves the delivery of agents withestablished HSV-immune reactivity (such as effector cells or antibodies)that can directly or indirectly mediate therapeutic effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper lymphocytes), killer cells(such as Natural Killer cells and lymphokine-activated killer cells), Bcells and antigen-presenting cells (such as dendritic cells andmacrophages) expressing a polypeptide provided herein. T cell receptorsand antibody receptors specific for the polypeptides recited herein maybe cloned, expressed and transferred into other vectors or effectorcells for adoptive immunotherapy. The polypeptides provided herein mayalso be used to generate antibodies or anti-idiotypic antibodies (asdescribed above and in U.S. Pat. No. 4,918,164) for passiveimmunotherapy.

[0339] Effector cells may generally be obtained in sufficient quantitiesfor adoptive immunotherapy by growth in vitro, as described herein.Culture conditions for expanding single antigen-specific effector cellsto several billion in number with retention of antigen recognition invivo are well known in the art. Such in vitro culture conditionstypically use intermittent stimulation with antigen, often in thepresence of cytokines (such as IL-2) and non-dividing feeder cells. Asnoted above, immunoreactive polypeptides as provided herein may be usedto rapidly expand antigen-specific T cell cultures in order to generatea sufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

[0340] Alternatively, a vector expressing a polypeptide recited hereinmay be introduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary or intraperitoneal.

[0341] Routes and frequency of administration of the therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, but may be readily established using standardtechniques. In one embodiment, between 1 and about 10 doses may beadministered over a 52 week period. In another embodiment, about 6 dosesare administered, at intervals of about 1 month, and boostervaccinations are typically be given periodically thereafter. Alternateprotocols may be appropriate for individual patients.

[0342] A suitable dose is an amount of a compound that, whenadministered as described above, is capable of promoting an anti-HSVimmune response, and is preferably at least 10-50% above the basal(i.e., untreated) level. Such response can be monitored, for example, bymeasuring the anti-HSV antibodies in a patient. Such vaccines shouldalso be capable of causing an immune response that leads to an improvedclinical outcome (e.g., more frequent remissions, complete or partial orlonger disease-free survival) in vaccinated patients as compared tonon-vaccinated patients. In general, for pharmaceutical compositions andvaccines comprising one or more polypeptides, the amount of eachpolypeptide present in a dose ranges from about 25 μg to 5 mg per kg ofhost. Suitable dose sizes will vary with the size of the patient, butwill typically range from about 0.1 mL to about 5 mL.

[0343] In general, an appropriate dosage and treatment regimen providesthe active compound(s) in an amount sufficient to provide therapeuticand/or prophylactic benefit. Such a response can be monitored byestablishing an improved clinical outcome (e.g., more frequentremissions, complete or partial, or longer disease-free survival) intreated patients as compared to non-treated patients. Increases inpreexisting immune responses to a HSV protein may correlate with animproved clinical outcome. Such immune responses may generally beevaluated using standard proliferation, cytotoxicity or cytokine assays,which may be performed using samples obtained from a patient before andafter treatment.

[0344] HSV Detection and Diagnosis

[0345] In general, HSV may be detected in a patient based on thepresence of one or more HSV proteins and/or polynucleotides encodingsuch proteins in a biological sample (for example, blood, sera, sputumurine and/or other appropriate tissue) obtained from the patient. Inother words, such proteins may be used as markers to indicate thepresence or absence of HSV in a patient. The binding agents providedherein generally permit detection of the level of antigen that binds tothe agent in the biological sample. Polynucleotide primers and probesmay be used to detect the level of mRNA encoding a HSV protein, which isalso indicative of the presence or absence of HSV infection.

[0346] There are a variety of assay formats known to those of ordinaryskill in the art for using a binding agent to detect polypeptide markersin a sample. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In general, the presence orabsence of HSV in a patient may be determined by contacting a biologicalsample obtained from a patient with a binding agent and detecting in thesample a level of polypeptide that binds to the binding agent.

[0347] In a preferred embodiment, the assay involves the use of bindingagent immobilized on a solid support to bind to and remove thepolypeptide from the remainder of the sample. The bound polypeptide maythen be detected using a detection reagent that contains a reportergroup and specifically binds to the binding agent/polypeptide complex.Such detection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length HSV proteins and portions thereof to which the binding agentbinds, as described above.

[0348] The solid support may be any material known to those of ordinaryskill in the art to which the protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

[0349] Covalent attachment of binding agent to a solid support maygenerally be achieved by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the binding agent. For example,the binding agent may be covalently attached to supports having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe binding partner (see, e.g., Pierce Immunotechnology Catalog andHandbook, 1991, at A12-A13).

[0350] In certain embodiments, the assay is a two-antibody sandwichassay. This assay may be performed by first contacting an antibody thathas been immobilized on a solid support, commonly the well of amicrotiter plate, with the sample, such that polypeptides within thesample are allowed to bind to the immobilized antibody. Unbound sampleis then removed from the immobilized polypeptide-antibody complexes anda detection reagent (preferably a second antibody capable of binding toa different site on the polypeptide) containing a reporter group isadded. The amount of detection reagent that remains bound to the solidsupport is then determined using a method appropriate for the specificreporter group.

[0351] More specifically, once the antibody is immobilized on thesupport as described above, the remaining protein binding sites on thesupport are typically blocked. Any suitable blocking agent known tothose of ordinary skill in the art, such as bovine serum albumin orTween 20™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibodyis then incubated with the sample, and polypeptide is allowed to bind tothe antibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with an HSV infection. Preferably, thecontact time is sufficient to achieve a level of binding that is atleast about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

[0352] Unbound sample may then be removed by washing the solid supportwith an appropriate buffer, such as PBS containing 0.1% Tween 2OTM. Thesecond antibody, which contains a reporter group, may then be added tothe solid support. Preferred reporter groups include those groupsrecited above.

[0353] The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

[0354] To determine the presence or absence of HSV, the signal detectedfrom the reporter group that remains bound to the solid support isgenerally compared to a signal that corresponds to a predeterminedcut-off value. In one embodiment, the cut-off value for the detection ofHSV is the average mean signal obtained when the immobilized antibody isincubated with samples from patients without HSV. In an alternateembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive.

[0355] In a related embodiment, the assay is performed in a flow-throughor strip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of HSV. Typically, the concentration of second binding agent atthat site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

[0356] Of course, numerous other assay protocols exist that are suitablefor use with the HSV proteins or binding agents of the presentinvention. The above descriptions are intended to be exemplary only. Forexample, it will be apparent to those of ordinary skill in the art thatthe above protocols may be readily modified to use HSV polypeptides todetect antibodies that bind to such polypeptides in a biological sample.The detection of such protein-specific antibodies can allow for theidentification of HSV infection.

[0357] HSV infection may also, or alternatively, be detected based onthe presence of T cells that specifically react with a HSV protein in abiological sample. Within certain methods, a biological samplecomprising CD4⁺ and/or CD8⁺ T cells isolated from a patient is incubatedwith a HSV polypeptide, a polynucleotide encoding such a polypeptideand/or an APC that expresses at least an immunogenic portion of such apolypeptide, and the presence or absence of specific activation of the Tcells is detected. Suitable biological samples include, but are notlimited to, isolated T cells. For example, T cells may be isolated froma patient by routine techniques (such as by Ficoll/Hypaque densitygradient centrifugation of peripheral blood lymphocytes). T cells may beincubated in vitro for about 2-9 days (typically about 4 days) at 37° C.with polypeptide (e.g., 5-25 μg/ml). It may be desirable to incubateanother aliquot of a T cell sample in the absence of HSV polypeptide toserve as a control. For CD4⁺ T cells, activation is preferably detectedby evaluating proliferation of the T cells. For CD8⁺ T cells, activationis preferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of HSV in the patient.

[0358] As noted above, HSV infection may also, or alternatively, bedetected based on the level of mRNA encoding a HSV protein in abiological sample. For example, at least two oligonucleotide primers maybe employed in a polymerase chain reaction (PCR) based assay to amplifya portion of a HSV cDNA derived from a biological sample, wherein atleast one of the oligonucleotide primers is specific for (i.e.,hybridizes to) a polynucleotide encoding the HSV protein. The amplifiedCDNA is then separated and detected using techniques well known in theart, such as gel electrophoresis. Similarly, oligonucleotide probes thatspecifically hybridize to a polynucleotide encoding a HSV protein may beused in a hybridization assay to detect the presence of polynucleotideencoding the HSV protein in a biological sample.

[0359] To permit hybridization under assay conditions, oligonucleotideprimers and probes should comprise an oligonucleotide sequence that hasat least about 60%, preferably at least about 75% and more preferably atleast about 90%, identity to a portion of a polynucleotide encoding aHSV protein that is at least 10 nucleotides, and preferably at least 20nucleotides, in length. Preferably, oligonucleotide primers and/orprobes hybridize to a polynucleotide encoding a polypeptide describedherein under moderately stringent conditions, as defined above.Oligonucleotide primers and/or probes which may be usefully employed inthe diagnostic methods described herein preferably are at least 10-40nucleotides in length. In a preferred embodiment, the oligonucleotideprimers comprise at least 10 contiguous nucleotides, more preferably atleast 15 contiguous nucleotides, of a DNA molecule having a sequencedisclosed herein. Techniques for both PCR based assays and hybridizationassays are well known in the art (see, for example, Mullis et al., ColdSpring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCRTechnology, Stockton Press, NY, 1989).

[0360] One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not infected with HSV. The amplification reaction may beperformed on several dilutions of cDNA, for example spanning two ordersof magnitude.

[0361] As noted above, to improve sensitivity, multiple HSV proteinmarkers may be assayed within a given sample. It will be apparent thatbinding agents specific for different HSV polypeptides may be combinedwithin a single assay. Further, multiple primers or probes may be usedconcurrently. The selection of HSV protein markers may be based onroutine experiments to determine combinations that results in optimalsensitivity. In addition, or alternatively, assays for HSV proteinsprovided herein may be combined with assays for other known HSVantigens.

[0362] The present invention further provides kits for use within any ofthe above diagnostic and/or therapeutic methods. Such kits typicallycomprise two or more components necessary for performing a diagnosticand/or therapeutic assay and will further comprise instructions for theuse of said kit. Components may be compounds, reagents, containersand/or equipment. For example, one container within a diagnostic kit maycontain a monoclonal antibody or fragment thereof that specificallybinds to a HSV protein. Such antibodies or fragments may be providedattached to a support material, as described above. One or moreadditional containers may enclose elements, such as reagents or buffers,to be used in the assay. Such kits may also, or alternatively, contain adetection reagent as described above that contains a reporter groupsuitable for direct or indirect detection of antibody binding.

[0363] Alternatively, a kit may be designed to detect the level of mRNAencoding a HSV protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a HSV protein. Suchan oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding aHSV protein.

[0364] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLE 1 Identification of HSV-2 Antigens

[0365] The following examples are presented to illustrate certainembodiments of the present invention and to assist one of ordinary skillin making and using the same. The examples are not intended in any wayto otherwise limit the scope of the invention.

[0366] Source of HSV-2 Positive Donors:

[0367] Lymphocytes were obtained from two types of donors: Group A)seropositive donors with unknown clinical status, and Group B)seropositive donors with well characterized clinical status (viralshedding and ano-genital lesion recurrences).

[0368] Group A: Blood samples (50 ml) were obtained from 13 potentialdonors. No information regarding clinical history of HSV-2 infection wasrequested. The blood was screened for serum antibody against HSV-1 andHSV-2 by Western blot. PBMCs were also screened for specificproliferative T cell responses to HSV-1 and HSV-2 lysate antigens (ABI;Columbia, Md.). Three donors (AD104, AD116, and AD120) were positive forHSV-2 serum antibody and their PBMCs specifically proliferated inresponse to HSV-2 antigen. Leukopheresis PBMC were collected from thesedonors and cryopreserved in liquid nitrogen.

[0369] Group B: Ano-genital lesion biopisies were obtained from donorsDK21318 and JR5032. Lesion biopsy lymphocytes were expanded in vitrowith IL-2 and PHA in the presence of 50 uM acyclovir and subsequentlycryopreserved in liquid nitrogen. Typically 5×10⁶ to 5×10⁷ lymphocytesare obtained after two weeks. Autologous PBMC were also collected fromthe blood of DK23 18 and JR5032 and cryopreserved in liquid nitrogen.

[0370] Generation of CD4⁺ T Cell Lines:

[0371] Cryopreserved PBMCs or lesion-biopsy lymphocytes were thawed andstimulated in vitro with 1 ug/ml HSV-2 antigen (ABI) in RPMI 1640+10%human serum+10 ng/ml IL-7. Irradiated autologous PBMC were added asantigen presenting cells for the lesion biopsy lymphocytes only.Recombinant IL-2 (1 ng/ml) was added on days 1 and 4. The cells wereharvested, washed, and replated in fresh medium containing IL-2 and IL-7on day 7. Recombinant IL-2 was again added on day 10. The T cells wereharvested, washed, and restimulated in vitro with HSV-2 antigen plusirradiated autologous PBMCin the same manner on day 14 of culture. The Tcell lines were cryopreserved at 1×10⁷ cells/vial in liquid nitrogen onday 11-12 of the secondary stimulation. After thawing, the cryopreservedT cells retained the ability to specifically proliferate to HSV-2antigen in vitro. These T cells were subsequently used to screen HSV-2gene-fragment expression cloning libraries prepared in E. coli, asdescribed below.

[0372] Preparation of HSV-2 (333) DNA:

[0373] HSV-2 strain 333 virus was grown in Vero cells cultured in rollerbottles in 200 ml/bottle of Medium 199 (Gibco)+5% FCS. Vero cells aretransformed African green monkey fibroblast-like cells that wereobtained from ATCC (Cat. # CCL-81). Near-confluence Vero cells (10roller bottles) were infected with HSV-2 strain 333 virus at an MOI of0.01 in 50 ml/bottle of Medium 199+1% FCS. Cells and medium wereharvested from the roller bottles and the cells pelleted. Thesupernatant was saved on ice and the cell pellets were resuspended infresh Medium 199±1% FCS and lysed by 6 cycles of freezing/thawing. Thecell debris in the lysates was pelleted and the supernatant pooled withthe saved culture supernatant. Virus was pelleted from the pooledsupernatants by ultracentrifugation (12,000 g, 2 hours, 4° C.) andresuspended in 2 ml of fresh Medium 199+1% FCS. The virus was furtherpurified on a 5-15% linear Ficoll gradient by ultracentrifugation(19,000 g, 2 hours, 4° C.) as previously described (Chapter 10:Herpessimplex virus vectors of Molecular Virology: A Practical Approach(1993); Authors: F. J. Rixon and J. McClaughlan, Editors: A. J. Davisonand R. M. Elliott; Publisher: Oxford University Press, Inc, New York,N.Y.). The HSV-2 virus-containing band was extracted from the gradient,diluted 1 0-fold with Medium 199, and the virus pelleted byultracentrifugation at 19,000 g for 4 hours at 4° C. The virus pelletwas recovered and resuspended in 10 ml of Tris/EDTA (TE) buffer. Intactvirions were treated with DNAse and RNAse to remove cellular DNA andRNA. The enzymes were then inactivated by addition of EDTA andincubation at 65° C. DNA was prepared from the gradient-purified virusby lysis of the viral particles with SDS in the presence of EDTA,followed by phenol/chlorform extraction to purify the genomic viral DNA.HSV-2 DNA was precipitated with EtOH and the DNA pellet was dried andresuspended in 1 ml of Tris/EDTA buffer. The concentration and purity ofthe DNA was determined by reading the OD 260 and OD 280 on a UVspectrophotometer. Genomic DNA prepared in this manner was used forconstruction of an HSV-2 genomic fragment expression library in E. coli.

[0374] Construction of HSV-2 DNA Fragment Libraries in the pET17bVector:

[0375] The HSV2-I library was constructed as follows. DNA fragments weregenerated by sonicating genomic HSV-2 DNA for 4 seconds at 15% outputwith a Fisher “60 SonicDismembrator” (Fisher). The sonicated DNA wasthen precipitated, pelleted, and resuspended in 11 uL TE buffer. Theapproximate size of the DNA fragments was measured by agarose gelelectropheresis of 1 uL of the fragmented HSV-2 genomic DNA vs. 1.5 ugunsonicated material. The average size of the DNA fragments wasdetermined to be approx. 500 bp when visualized after ethidium bromidestaining of the gel. Incomplete DNA fragment ends were filled in(blunted) usingT4 DNA polymerase. EcoRl adapters were then ligated tothe blunt ends of the DNA fragments using T4 DNA ligase. The DNA wasthen kinased using T4 Polynucleotide Kinase, purified using a manuallyloaded column of S-400-HR Sephacryl (Sigma) and ligated into the pET17bexpression vector. The HSV2-II library was constructed in a similarfashion. The average size of inserts in this library was determined tobe approximately 1000 bp.

[0376] Generation of the HSV-2 Fragment Expression Library in E. coli.

[0377] The HSV2-I library was transformed into E. coli for preparationof glycerol stocks and testing of HSV-2 DNA insert representation. TheDNA was transformed into ElectroMAX DH10B E. coli (Gibco) in order toprepare a large quantity of HSV-2/pET17b library DNA. Transformedbacteria were grown up on 3 LB/Ampicillin plates (approx. 750CFU/plate), a small subset of colonies were picked for sequencing of DNAinserts, and the remaining bacteria from each plate collected as a poolfor preparation of plasmid DNA. These pools were named HSV-2 Pools 9, 10and 11. Glycerol stocks of a portion of these bacterial pools werestored at −80° C. Plasmids were purified from the remainder of thepools. Equal quantities of plasmid DNA from each of the 3 pools wascombined to make a single pool of plasmid DNA. The tranformationefficiency of the pooled DNA was empirically determined using JM109(DE3)E. coli bacteria. JM1 09(DE3) bacteria were then transformed with anamount of the final pool of library DNA that was expected to yield 15colony-forming units (CFU) per plate. The transformed bacteria were thenplated on 100 LB/amp plates. Twenty CFU (on average) were actuallyobserved on each of the 100 plates; therefore the pool size of thisHSV-2 library was about 20 clones/pool. The bacterial colonies werecollected as a pool from each plate in approximately 800 ul/plate of LB+20% glycerol. Each pool was distributed equally (200 ul/well) amongfour 96-well U-bottom plates and these “master stock” plates were storedat −80° C. The size of this HSV-2 gene-fragment library (hereafterreferred to as HSV2I) was therefore 96 pools of 20 clones/pool. PlasmidDNA was prepared from 20 randomly picked colonies and the insertssequenced. Approximately 15% (3/20) contained HSV-2 DNA as insert, 80%(16/20) contained non-HSV-2 DNA (E. coli or Vero cell DNA), and 5%(1/20) contained no insert DNA. The HSV2-II DNA library was transformedinto E. coli and random colonies analyzed in a similar manner. Relevantdifferences in the construction of library HSV2-II included thetransformation of the HSV-2/pET17b ligation product into NovaBlue(Novagen) chemically competent E. coli instead of using electroporationfor preparation of a larger quantity of plasmid for pooling andtransformation into JM109(DE3) bacteria for empirical evaluation.Additionally, plasmid DNA was prepared from 10 pools averaging 160colonies/plate. These 10 plasmid pools were combined in an equivalentfashion (normalized based on spectrophotometer readings) into one poolfor transformation into JM109(DE3) as per previously, yielding anaverage of 20 colonies(clones)/plate for harvesting into glycerol stockpools as before. Approximately 25% contained HSV-2 DNA as insert, withthe remaining 75% containing E. coli DNA as insert.

[0378] Induction of the HSV-2 Fragment Expression Library for Screeningwith Human CD4⁺ T Cells.

[0379] One of the master HSV2I library 96-well plates was thawed at roomtemperature. An aliquot (20 uL) was transferred from each well to a new96 well plate containing 180 uL/well of LB medium+ampicillin. Thebacteria were grown up overnight and then 40 ul transferred into two new96-well plates containing 160 uL 2xYT medium+ampicillin. The bacteriawere grown for 1 hr.15 min at 37° C. Protein expression was then inducedby addition of IPTG to 200 mM. The bacteria were cultured for anadditional 3 hrs. One of these plates was used to obtainspectrophotometer readings to normalize bacterial numbers/well. Thesecond, normalized plate was used for screening with CD4⁺ T cells afterpelleting the bacteria (approx. 2×10⁷/well) and removing thesupernatants. The HSV2-II library was grown and induced in a similarfashion.

[0380] Preparation of Autologous Dendritic APC's:

[0381] Dendritic cells (DCs) were generated by culture ofplastic-adherent donor cells (derived from 1×10⁸ PBMC) in 6 well plates(Costar 3506) in RPMI 1640+10% of a 1:1 mix of FCS:HS+10 ng/ml GM-CSF+10 ng/ml IL-4 at 37° C. Non-adherent DCs were collected from plates onday 6 of culture and irradiated with 3300 Rads. The DCs were then platedat 1×10⁴/well in flat-bottom 96-well plates (Costar 3596) and culturedovernight at 37° C. The following day, the DCs were pulsed with theinduced HSV2-I or HSV2-II library pools by resuspending the bacterialpellets in 200 ul RPMI 1640+10%FCS without antibiotics and transferring10 ul/well to the wells containing the DCs in 190 ul of the same mediumwithout antibiotics. The DCs and bacteria were co-cultured for 90minutes at 37° C. The DCs were then washed and resuspended in 100ul/well RPMI 1640+10% HS +L-glut. +50 ug/ml gentamicin antibiotic.

[0382] Preparation of Responder T cells:

[0383] Cryopreserved CD4+ T cell lines were thawed 5 days before use andcultured at 3 7° C. in RPMI 1640+10% HS+1 ng/ml IL-2+10 ng/ml IL-7.After 2 days, the medium was replaced with fresh medium without IL-2 andIL-7.

[0384] Primary Screening of the HSV2 Libraries:

[0385] The T cells were resuspended in fresh RPMI 1640+10% HS and addedat 2×10⁴/well to the plates containing the E. coli-pulsed autologousDC's. After 3 days, 100 ul/well of supernatant was removed andtransferred to new 96 well plates. Half of the supernatant wassubsequently tested for IFN-gamma content by ELISA and the remainder wasstored at −20° C. The T cells were then pulsed with 1 uCi/well of[3H]-Thymidine (Amersham/Pharmacia; Piscataway, N.J.) for about 8 hoursat 37° C. The 3H-pulsed cells were then harvested onto UniFilter GF/Cplates (Packard; Downers Grove, Ill.) and the CPM of [3H]-incorporatedsubsequently measured using a scintillation counter (Top-Count;Packard). ELISA assays were performed on cell supernatants following astandard cytokine-capture ELISA protocol for human IFN-g.

[0386] From the HSV2-I library screening with T cells from D104, wellsHSV2I_H10 and HSV2I_H12, for which both CPM and IFN-g levels weresignificantly above background, were scored as positive.

[0387] Breakdown of Positive HSV2I Library Pools:

[0388] The positive wells (HSV2I_H10 and HSV2I_H12) from the initialCD4+ T cell screening experiment were grown up again from the masterglycerol stock plate. Forty-eight sub-clones from each pool wererandomly picked, grown up and IPTG-induced as described previously. Thesubclones were screened against the AD104 CD4+ T cell line as describedabove. A clone (HSV2I_H12A12) from the HSV2I_H12 pool breakdown scoredpositive. This positive result was verified in a second AD 104 CD4+ Tcell assay.

[0389] Identification of UL39 as a CD4+ T Cell Antigen:

[0390] The positive clone (HSV2I_H12A12) was subcloned and 10 clonespicked for restriction digest analysis with EcoRI NB#675 pg. 34. All 10clones contained DNA insert of the same size (approximately 900 bp inlength). Three of these clones (HSV2I_H12A12_(—)1, 7, and 8) were chosenfor sequencing and all contained identical insert sequences at both the5′ and 3′ ends of the inserts. The DNA sequence of the insert is setforth in SEQ ID NO: 1, and contains an open reading frame set forth inSEQ ID NO:2. The insert sequence was compared to the complete genomicsequence of HSV-2 strain HG52 (NCBI site, Accession #Z86099) and thesequence was determined have a high degree of homology to UL39 (a.k.a.ICP6), the large subunit (140 kD) of the HSV ribonucleotide reductase,the sequence of which is set forth in SEQ ID NO:3. The insert sequenceset forth in SEQ ID NO: 1 spans nucleotides 876-1690 of the UL39 openreading frame (3,432 bp) and encodes the amino acid sequence set forthin SEQ ID NO:2, which has a high degree of homology to amino acids292-563 of UL39 (full length=1143 aa).

[0391] Identification of US8A, US3/US4, UL15, UL18, UL27 and UL46 asCD4+ T Cell Antigens:

[0392] In a manner essentially identical to that described above for theidentification of UL39 as a T cell antigen, an additional HSV2 genefragment expression cloning library, referred to as HSV2-II, wasprepared, expressed in E. coli, and screened with donor T cells.

[0393] Screening the HSV2-II library with T cells from donor AD 116identified the clone HSV2II_US8AfragD6.B_B11_T7Trc.seq, determined tohave an insert sequence set forth in SEQ ID NO:4, encoding open readingframes having amino acid sequences set forth in SEQ ID NO:5 and 6, withthe sequence of SEQ ID NO:5 having a high degree of homology with theHSV2 US8A protein, the sequence of which is set forth in SEQ ID NO:7.

[0394] In addition, screening the HSV2-II library with T cells fromdonor AD104 identified the following clone inserts:

[0395] SEQ ID NO:8, corresponding to clone HSV2II_US3/IUS4fragF10B3_T7Trc.seq, containing a potential open reading frame having anamino acid sequence set forth in SEQ ID NO: 10;

[0396] SEQ ID NO:9, corresponding to clone HSV2II_US3/US4 fragF10B3T7P.seq, containing an open reading frame having an amino acid sequenceset forth in SEQ ID NO: 11, sharing a high degree of homology with theHSV-2 US3 protein (SEQ ID NO: 12);

[0397] SEQ ID NO:13, corresponding to cloneHSV2II_UL46fragF11F5_T7Trc.seq, containing an open reading frame havingan amino acid sequence set forth in SEQ ID NO: 14, sharing a high degreeof homology with the HSV-2 UL46 protein (SEQ ID NO: 15);

[0398] SEQ ID NO:16, corresponding to cloneHSV2II_UL27frag-H2C7_T7Trc.seq, containing an open reading frame havingan amino acid sequence set forth in SEQ ID NO: 17, sharing a high degreeof homology with the HSV-2 UL27 protein (SEQ ID NO: 18);

[0399] SEQ ID NO:19, corresponding to clone HSV2II_UL18fragF10A11rc.seq,containing open reading frames having amino acid sequences set forth inSEQ ID NO:20, 21 and 22, with SEQ ID NO:22 sharing a high degree ofhomology with the HSV-2 UL18 protein (SEQ ID NO: 23); and

[0400] SEQ ID NO:24, corresponding to cloneHSV2II_UL15fragF10A12_rc.seq, containing an open reading frame having anamino acid sequence set forth in SEQ ID NO: 25, sharing a high degree ofhomology with the HSV-2 UL15 protein (SEQ ID NO: 26).

EXAMPLE 2 Identification of HSV-2 Antigens

[0401] CD4⁺ T cells from AD104 were found to recognize inserts fromclones HSV2II_UL46fragF11F5_T7Trc.seq (SEQ ID NO: 13) andHSV2II_UL18frgaF10A1_rc.seq (SEQ ID NO: 19) as described in detail inExample 1. The sequences from these clones share a high degree ofhomology to the HSV2-I genes, UL46 (SEQ ID NO: 15) and UL18 (SEQ IDNO:23), respectively. Therefore to further characterize the epitopesrecognized by these T cells, overlapping 15-mer peptides were madeacross the clone insert fragments of UL18 and UL46. Peptide recognitionby AD104's CD4⁺ T cells was tested in a 48 hour IFN-g ELISPOT assay.ELISPOTS were performed by adding 1×10⁴ autologous EBV-transformed Bcells (LCL) or DCs per well in 96 well ELISPOT plates. 2×10⁴ AD104 CD4+T cells from AD104's line were added per well with 5 μg/ml of the HSV2peptides. AD104 CD4+ T cells recognized peptides 20 and 21 (SEQ ID NO:32 and 33) of UL18, and peptides 1, 4, 9, 10, and 20 of UL46 (SEQ ID NO:27-31).

EXAMPLE 3 Identification of HSV-2 Antigens

[0402] CD4+ T cell lines were generated from DK23 18 and JR5032lesion-biopsy. The CD4+ lymphocytes were stimulated twice in vitro onirradiated autologous PBMC and HSV2 antigen as described in example 1.The lines were tested for their antigen specificity as described inexample 1 and cryopreserved. The CD4+ T cell lines were screened againstthe HSV2-II expression-cloning library generated in Example 1.

[0403] DK2318 was shown to react with clones C12 and G10. Clone C12 wasdetermined to have an insert sequence set forth in SEQ ID NO:36. Thisinsert was found to have sequence homology with fragments of 2 HSV-IIgenes, nucleotides 723-1311 of UL23 and nucleotides 1-852 of UL22. Thesesequences correspond to amino acids 241-376 of UL23 as set forth in SEQID NO:40 and amino acids 1-284 as set forth in SEQ ID NO:41. The DNAsequence of SEQ ID NO:36 was searched against public databases includingGenbank and shown to have a high degree of sequence homology to the HSV2genes UL23 and UL22 set forth in SEQ ID NO:37 and 38 respectively. Theprotein sequences encoded by SEQ ID NO:37 and 38 are set forth in SEQ IDNO:39 and 45. Clone G10 was determined to have an insert sequence whichis set forth in SEQ ID NO:48, encoding open reading frames having anamino acid sequence set forth in SEQ ID NO:50, with the sequence of SEQID NO:48 having a high degree of sequence homology with HSV2 UL37, thesequence of which is set forth in SEQ ID NO:49, encoding open readingframes having the amino acid sequences set forth in SEQ ID NO:51.DK2318's CD4+ T cell line was screened against overlapping 15 merscovering the UL23 protein. DK2318's CD4 line was shown to react againstthree UL23 specific peptides (SEQ ID NO:41-43) suggesting that UL23 is atarget.

[0404] The CD4+ T cell line generated from JR5032 was found to reactwith clone E9 which contained an insert sequence set forth in SEQ ID NO:34, encoding open reading frames having amino acid sequences set forthin SEQ ID NO: 46, with SEQ ID NO: 34 having a high degree of sequencehomology with HSV2 RL2 (also referred to as ICPO), the sequence of whichis set forth in SEQ ID NO:35, encoding an open reading frame having theamino acid sequences set forth in SEQ ID NO:47.

EXAMPLE 4 Characterization of CD4 Clones F11F5 and G10A9

[0405] Examples 2 and 3 describe the generation of CD4 T cell lines fromdonors AD104 and DK2313 which were screened against cDNA librariesgenerated using the HSV-2 333 strain. AD104 was found to react againstthe clone HSV2II_UL46fragF11F5. This insert was partially sequenced withthe sequence being disclosed in SEQ ID NO:13. Full length sequencing ofthe insert revealed that it encoded a fragment of UL46 which was derivedfrom the HSV-2 333 strain. The DNA and amino acid sequences from thisinsert are disclosed in SEQ ID NO:52 and 54, respectively.

[0406] DK2312 was found to react against the clone G10. This insert waspartially sequenced and the sequence was disclosed in SEQ ID NO:48. Fulllength sequencing revealed that it encoded a fragment of UL37 which wasderived from the HSV-2 333 strain. The DNA and amino acid sequences fromthis insert are disclosed in SEQ ID NO:53 and 55, respectively.

EXAMPLE 5 Identification of CD8-specific Immunoreactive Peptides Derivedfrom HSV-2

[0407] Peripheral blood mononuclear cells were obtained from the normaldonors AD104, ADI 16, AD120, and D477. These donors were HLA typed usinglow-resolution DNA-typing methodology and the results are presented inTable 2. TABLE 2 DONOR AD104 AD116 AD120 D477 HLA-A 24, 33 0206, 240211, 3303 0201, 2501 HLA-B 45, 58 0702, 35 1505, 4403 1501, 5101 HLA-C01, 0302 0702, 1203 0303, 0706 0304, 12

[0408] In order to determine which epitopes of HSV-2 wereimmunoreactive, synthetic peptides were synthesized. These peptides were15 amino acids in length overlapping by 11 amino acids. The peptideswere synthesized across the following regions of the following HSV-2genes: UL15 (aa 600-734), UL18 (aa 1-110), UL23 (aa 241-376), UL46 (aa617-722), US3 (aa125-276), and US8A (aa 83-146).

[0409] CD8⁺ T cells were purified from the PBMC of each of the donorsdescribed above using negative selection. The purified CD8⁺ T cells werethen tested for their reactivity against the HSV-2 specific peptides.Co-cultures containing 2×10⁵ CD8+ T cells, 1×10⁴ autologous dendriticcells and 10 μg/ml of a peptide pool (on average containing 10peptides/pool) were established in 96 well ELISPOT plates that had beenpre-coated with anti-human IFN-γ antibody (1D1K: mAbTech). After 24hours, the ELISPOT plates were developed using a standard protocol wellknown to one of skill in the art. The number of spots per well were thencounted using an automated video microscopy ELISPOT plate reader. CD8+ Tcells from donors demonstrating a positive response against a peptidepool were then subsequently tested against the individual peptides inthat pool in a second ELISPOT assay. The results of peptide reactivityare presented in Table 3. TABLE 3 Peptide # Donor HSV-2 Gene (amino acidnumbering) SEQ ID NO AD104 US3 #33 (262-276) 63 AD116 UL15 #23 (688-702)56 #30 (716-730) 57 UL23  #7 (265-279) 58 UL46  #2 (621-635) 59  #8(645-659) 60  #9 (649-663) 61 #11 (657-671) 62 US8A  #5 (99-113) 64AD120 UL46 Peptides: #1-12 — D477 UL18 Peptides: #1-12 — UL23 Peptides:#1-20 — UL46 Peptides: #1-12 —

[0410] Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 64 <210> SEQ ID NO 1<211> LENGTH: 815 <212> TYPE: DNA <213> ORGANISM: Herpes simplex virus<400> SEQUENCE: 1 ccacgccgcc gcaccccagg cggacgtggc gccggttctg gacagccagcccactgtggg 60 aacggacccc ggctacccag tccccctaga actcacgccc gagaacgcggaggcggtggc 120 gcggtttctg ggggacgccg tcgaccgcga gcccgcgctc atgctggagtacttctgtcg 180 gtgcgcccgc gaggagagca agcgcgtgcc cccacgaacc ttcggcagcgccccccgcct 240 cacggaggac gactttgggc tcctgaacta cgcgctcgct gagatgcgacgcctgtgcct 300 ggaccttccc ccggtccccc ccaacgcata cacgccctat catctgagggagtatgcgac 360 gcggctggtt aacgggttca aacccctggt gcggcggtcc gcccgcctgtatcgcatcct 420 ggggattctg gttcacctgc gcatccgtac ccgggaggcc tcctttgaggaatggatgcg 480 ctccaaggag gtggacctgg acttcgggct gacggaaagg cttcgcgaacacgaggccca 540 gctaatgatc ctggcccagg ccctgaaccc ctacgactgt ctgatccacagcaccccgaa 600 cacgctcgtc gagcgggggc tgcagtcggc gctgaagtac gaagagttttacctcaagcg 660 cttcggcggg cactacatgg agtccgtctt ccagatgtac acccgcatcgccgggttcct 720 ggcgtgccgg gcgacccgcg gcatgcgcca catcgccctg gggcgacaggggtcgtggtg 780 ggaaatgttc aagttctttt tccaccgcct ctacg 815 <210> SEQ IDNO 2 <211> LENGTH: 271 <212> TYPE: PRT <213> ORGANISM: Herpes simplexvirus <400> SEQUENCE: 2 His Ala Ala Ala Pro Gln Ala Asp Val Ala Pro ValLeu Asp Ser Gln 1 5 10 15 Pro Thr Val Gly Thr Asp Pro Gly Tyr Pro ValPro Leu Glu Leu Thr 20 25 30 Pro Glu Asn Ala Glu Ala Val Ala Arg Phe LeuGly Asp Ala Val Asp 35 40 45 Arg Glu Pro Ala Leu Met Leu Glu Tyr Phe CysArg Cys Ala Arg Glu 50 55 60 Glu Ser Lys Arg Val Pro Pro Arg Thr Phe GlySer Ala Pro Arg Leu 65 70 75 80 Thr Glu Asp Asp Phe Gly Leu Leu Asn TyrAla Leu Ala Glu Met Arg 85 90 95 Arg Leu Cys Leu Asp Leu Pro Pro Val ProPro Asn Ala Tyr Thr Pro 100 105 110 Tyr His Leu Arg Glu Tyr Ala Thr ArgLeu Val Asn Gly Phe Lys Pro 115 120 125 Leu Val Arg Arg Ser Ala Arg LeuTyr Arg Ile Leu Gly Ile Leu Val 130 135 140 His Leu Arg Ile Arg Thr ArgGlu Ala Ser Phe Glu Glu Trp Met Arg 145 150 155 160 Ser Lys Glu Val AspLeu Asp Phe Gly Leu Thr Glu Arg Leu Arg Glu 165 170 175 His Glu Ala GlnLeu Met Ile Leu Ala Gln Ala Leu Asn Pro Tyr Asp 180 185 190 Cys Leu IleHis Ser Thr Pro Asn Thr Leu Val Glu Arg Gly Leu Gln 195 200 205 Ser AlaLeu Lys Tyr Glu Glu Phe Tyr Leu Lys Arg Phe Gly Gly His 210 215 220 TyrMet Glu Ser Val Phe Gln Met Tyr Thr Arg Ile Ala Gly Phe Leu 225 230 235240 Ala Cys Arg Ala Thr Arg Gly Met Arg His Ile Ala Leu Gly Arg Gln 245250 255 Gly Ser Trp Trp Glu Met Phe Lys Phe Phe Phe His Arg Leu Tyr 260265 270 <210> SEQ ID NO 3 <211> LENGTH: 1142 <212> TYPE: PRT <213>ORGANISM: Herpes simplex virus <400> SEQUENCE: 3 Met Ala Asn Arg Pro AlaAla Ser Ala Leu Ala Gly Ala Arg Ser Pro 1 5 10 15 Ser Glu Arg Gln GluPro Arg Glu Pro Glu Val Ala Pro Pro Gly Gly 20 25 30 Asp His Val Phe CysArg Lys Val Ser Gly Val Met Val Leu Ser Ser 35 40 45 Asp Pro Pro Gly ProAla Ala Tyr Arg Ile Ser Asp Ser Ser Phe Val 50 55 60 Gln Cys Gly Ser AsnCys Ser Met Ile Ile Asp Gly Asp Val Ala Arg 65 70 75 80 Gly His Leu ArgAsp Leu Glu Gly Ala Thr Ser Thr Gly Ala Phe Val 85 90 95 Ala Ile Ser AsnVal Ala Ala Gly Gly Asp Gly Arg Thr Ala Val Val 100 105 110 Ala Leu GlyGly Thr Ser Gly Pro Ser Ala Thr Thr Ser Val Gly Thr 115 120 125 Gln ThrSer Gly Glu Phe Leu His Gly Asn Pro Arg Thr Pro Glu Pro 130 135 140 GlnGly Pro Gln Ala Val Pro Pro Pro Pro Pro Pro Pro Phe Pro Trp 145 150 155160 Gly His Glu Cys Cys Ala Arg Arg Asp Ala Arg Gly Gly Ala Glu Lys 165170 175 Asp Val Gly Ala Ala Glu Ser Trp Ser Asp Gly Pro Ser Ser Asp Ser180 185 190 Glu Thr Glu Asp Ser Asp Ser Ser Asp Glu Asp Thr Gly Ser GluThr 195 200 205 Leu Ser Arg Ser Ser Ser Ile Trp Ala Ala Gly Ala Thr AspAsp Asp 210 215 220 Asp Ser Asp Ser Asp Ser Arg Ser Asp Asp Ser Val GlnPro Asp Val 225 230 235 240 Val Val Arg Arg Arg Trp Ser Asp Gly Pro AlaPro Val Ala Phe Pro 245 250 255 Lys Pro Arg Arg Pro Gly Asp Ser Pro GlyAsn Pro Gly Leu Gly Ala 260 265 270 Gly Thr Gly Pro Gly Ser Ala Thr AspPro Arg Ala Ser Ala Asp Ser 275 280 285 Asp Ser Ala Ala His Ala Ala AlaPro Gln Ala Asp Val Ala Pro Val 290 295 300 Leu Asp Ser Gln Pro Thr ValGly Thr Asp Pro Gly Tyr Pro Val Pro 305 310 315 320 Leu Glu Leu Thr ProGlu Asn Ala Glu Ala Val Ala Arg Phe Leu Gly 325 330 335 Asp Ala Val AspArg Glu Pro Ala Leu Met Leu Glu Tyr Phe Cys Arg 340 345 350 Cys Ala ArgGlu Glu Ser Lys Arg Val Pro Pro Arg Thr Phe Gly Ser 355 360 365 Ala ProArg Leu Thr Glu Asp Asp Phe Gly Leu Leu Asn Tyr Ala Leu 370 375 380 AlaGlu Met Arg Arg Leu Cys Leu Asp Leu Pro Pro Val Pro Pro Asn 385 390 395400 Ala Tyr Thr Pro Tyr His Leu Arg Glu Tyr Ala Thr Arg Leu Val Asn 405410 415 Gly Phe Lys Pro Leu Val Arg Arg Ser Ala Arg Leu Tyr Arg Ile Leu420 425 430 Gly Val Leu Val His Leu Arg Ile Arg Thr Arg Glu Ala Ser PheGlu 435 440 445 Glu Trp Met Arg Ser Lys Glu Val Asp Leu Asp Phe Gly LeuThr Glu 450 455 460 Arg Leu Arg Glu His Glu Ala Gln Leu Met Ile Leu AlaGln Ala Leu 465 470 475 480 Asn Pro Tyr Asp Cys Leu Ile His Ser Thr ProAsn Thr Leu Val Glu 485 490 495 Arg Gly Leu Gln Ser Ala Leu Lys Tyr GluGlu Phe Tyr Leu Lys Arg 500 505 510 Phe Gly Gly His Tyr Met Glu Ser ValPhe Gln Met Tyr Thr Arg Ile 515 520 525 Ala Gly Phe Leu Ala Cys Arg AlaThr Arg Gly Met Arg His Ile Ala 530 535 540 Leu Gly Arg Gln Gly Ser TrpTrp Glu Met Phe Lys Phe Phe Phe His 545 550 555 560 Arg Leu Tyr Asp HisGln Ile Val Pro Ser Thr Pro Ala Met Leu Asn 565 570 575 Leu Gly Thr ArgAsn Tyr Tyr Thr Ser Ser Cys Tyr Leu Val Asn Pro 580 585 590 Gln Ala ThrThr Asn Gln Ala Thr Leu Arg Ala Ile Thr Gly Asn Val 595 600 605 Ser AlaIle Leu Ala Arg Asn Gly Gly Ile Gly Leu Cys Met Gln Ala 610 615 620 PheAsn Asp Ala Ser Pro Gly Thr Ala Ser Ile Met Pro Ala Leu Lys 625 630 635640 Val Leu Asp Ser Leu Val Ala Ala His Asn Lys Gln Ser Thr Arg Pro 645650 655 Thr Gly Ala Cys Val Tyr Leu Glu Pro Trp His Ser Asp Val Arg Ala660 665 670 Val Leu Arg Met Lys Gly Val Leu Ala Gly Glu Glu Ala Gln ArgCys 675 680 685 Asp Asn Ile Phe Ser Ala Leu Trp Met Pro Asp Leu Phe PheLys Arg 690 695 700 Leu Ile Arg His Leu Asp Gly Glu Lys Asn Val Thr TrpSer Leu Phe 705 710 715 720 Asp Arg Asp Thr Ser Met Ser Leu Ala Asp PheHis Gly Glu Glu Phe 725 730 735 Glu Lys Leu Tyr Glu His Leu Glu Ala MetGly Phe Gly Glu Thr Ile 740 745 750 Pro Ile Gln Asp Leu Ala Tyr Ala IleVal Arg Ser Ala Ala Thr Thr 755 760 765 Gly Ser Pro Phe Ile Met Phe LysAsp Ala Val Asn Arg His Tyr Ile 770 775 780 Tyr Asp Thr Gln Gly Ala AlaIle Ala Gly Ser Asn Leu Cys Thr Glu 785 790 795 800 Ile Val His Pro AlaSer Lys Arg Ser Ser Gly Val Cys Asn Leu Gly 805 810 815 Ser Val Asn LeuAla Arg Cys Val Ser Arg Gln Thr Phe Asp Phe Gly 820 825 830 Arg Leu ArgAsp Ala Val Gln Ala Cys Val Leu Met Val Asn Ile Met 835 840 845 Ile AspSer Thr Leu Gln Pro Thr Pro Gln Cys Thr Arg Gly Asn Asp 850 855 860 AsnLeu Arg Ser Met Gly Ile Gly Met Gln Gly Leu His Thr Ala Cys 865 870 875880 Leu Lys Met Gly Leu Asp Leu Glu Ser Ala Glu Phe Arg Asp Leu Asn 885890 895 Thr His Ile Ala Glu Val Met Leu Leu Ala Ala Met Lys Thr Ser Asn900 905 910 Ala Leu Cys Val Arg Gly Ala Arg Pro Phe Ser His Phe Lys ArgSer 915 920 925 Met Tyr Arg Ala Gly Arg Phe His Trp Glu Arg Phe Ser AsnAla Ser 930 935 940 Pro Arg Tyr Glu Gly Glu Trp Glu Met Leu Arg Gln SerMet Met Lys 945 950 955 960 His Gly Leu Arg Asn Ser Gln Phe Ile Ala LeuMet Pro Thr Ala Ala 965 970 975 Ser Ala Gln Ile Ser Asp Val Ser Glu GlyPhe Ala Pro Leu Phe Thr 980 985 990 Asn Leu Phe Ser Lys Val Thr Arg AspGly Glu Thr Leu Arg Pro Asn 995 1000 1005 Thr Leu Leu Leu Lys Glu LeuGlu Arg Thr Phe Gly Gly Lys Arg Leu 1010 1015 1020 Leu Asp Ala Met AspGly Leu Glu Ala Lys Gln Trp Ser Val Ala Gln 1025 1030 1035 1040 Ala LeuPro Cys Leu Asp Pro Ala His Pro Leu Arg Arg Phe Lys Thr 1045 1050 1055Ala Phe Asp Tyr Asp Gln Glu Leu Leu Ile Asp Leu Cys Ala Asp Arg 10601065 1070 Ala Pro Tyr Val Asp His Ser Gln Ser Met Thr Leu Tyr Val ThrGlu 1075 1080 1085 Lys Ala Asp Gly Thr Leu Pro Ala Ser Thr Leu Val ArgLeu Leu Val 1090 1095 1100 His Ala Tyr Lys Arg Gly Leu Lys Thr Gly MetTyr Tyr Cys Lys Val 1105 1110 1115 1120 Arg Lys Ala Thr Asn Ser Gly ValPhe Ala Gly Asp Asp Asn Ile Val 1125 1130 1135 Cys Thr Ser Cys Ala Leu1140 <210> SEQ ID NO 4 <211> LENGTH: 208 <212> TYPE: DNA <213> ORGANISM:Herpes simplex virus <400> SEQUENCE: 4 gcgccgcgcc cgcgtgccgc agaccacctcgcggcggctc ccccgcggcc tttcccgtgg 60 ccctccacgc cgtggacgcc ccctcccaattcgtcacctg gctcgccgtg cgctggctgc 120 ggggggcggt gggtctcggg gccgtcctgtgcgggattgc gttttacgtg acgtcaatcg 180 cccgaggcgc ataaaggtcc ggcggcca 208<210> SEQ ID NO 5 <211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM:Herpes simplex virus <400> SEQUENCE: 5 Gly Ala Ala Pro Ala Cys Arg ArgPro Pro Arg Gly Gly Ser Pro Ala 1 5 10 15 Ala Phe Pro Val Ala Leu HisAla Val Asp Ala Pro Ser Gln Phe Val 20 25 30 Thr Trp Leu Ala Val Arg TrpLeu Arg Gly Ala Val Gly Leu Gly Ala 35 40 45 Val Leu Cys Gly Ile Ala PheTyr Val Thr Ser Ile Ala Arg Gly Ala 50 55 60 <210> SEQ ID NO 6 <211>LENGTH: 70 <212> TYPE: PRT <213> ORGANISM: Herpes simplex virus <400>SEQUENCE: 6 Arg Arg Ala Arg Val Pro Gln Thr Thr Ser Arg Arg Leu Pro ArgGly 1 5 10 15 Leu Ser Arg Gly Pro Pro Arg Arg Gly Arg Pro Leu Pro IleArg His 20 25 30 Leu Ala Arg Arg Ala Leu Ala Ala Gly Gly Gly Gly Ser ArgGly Arg 35 40 45 Pro Val Arg Asp Cys Val Leu Arg Asp Val Asn Arg Pro ArgArg Ile 50 55 60 Lys Val Arg Arg Pro Ala 65 70 <210> SEQ ID NO 7 <211>LENGTH: 146 <212> TYPE: PRT <213> ORGANISM: Herpes simplex virus <400>SEQUENCE: 7 Met Asp Pro Ala Leu Arg Ser Tyr His Gln Arg Leu Arg Leu TyrThr 1 5 10 15 Pro Ile Ala Arg Gly Val Asn Leu Ala Ala Arg Ser Pro ProLeu Val 20 25 30 Arg Glu Ala Arg Ala Val Val Thr Pro Arg Pro Pro Ile ArgPro Ser 35 40 45 Ser Gly Lys Ala Ser Ser Asp Asp Ala Asp Val Gly Asp GluLeu Ile 50 55 60 Ala Ile Ala Asp Ala Arg Gly Asp Pro Pro Glu Thr Leu ProPro Gly 65 70 75 80 Ala Gly Gly Ala Ala Pro Ala Cys Arg Arg Pro Pro ArgGly Gly Ser 85 90 95 Pro Ala Ala Phe Pro Val Ala Leu His Ala Val Asp AlaPro Ser Gln 100 105 110 Phe Val Thr Trp Leu Ala Val Arg Trp Leu Arg GlyAla Val Gly Leu 115 120 125 Gly Ala Val Leu Cys Gly Ile Ala Phe Tyr ValThr Ser Ile Ala Arg 130 135 140 Gly Ala 145 <210> SEQ ID NO 8 <211>LENGTH: 137 <212> TYPE: DNA <213> ORGANISM: Herpes simplx virus <400>SEQUENCE: 8 ccccaccgcc cccccacagg cggcgcgtgc ggagggcggc ccgtgcgtccccccggtccc 60 cgcgggccgc ccgtggcgct cggtgccccc ggtatggtat tccgcccccaaccccgggtt 120 tcgtggcctg cgtttcc 137 <210> SEQ ID NO 9 <211> LENGTH:430 <212> TYPE: DNA <213> ORGANISM: Herpes simplex virus <400> SEQUENCE:9 atggaccggg aggcacttcg ggccatcagc cgcgggtgca agcccccttc gaccctggca 60aaactggtga ccgggctggg attcgcgatc cacggagcgc tcatcccggg gtcggagggg 120tgtgtctttg atagcagcca cccgaactac cctcatcggg taatcgtcaa ggcggggtgg 180tacgccagca cgaaccacga ggcgcggctg ctgagacgcc tgaaccaccc cgcgatccta 240cccctcctgg acctgcacgt cgtttctggg gtcacgtgtc tggtcctccc caagtatcac 300tgcgacctgt atacctatct gagcaagcgc ccgtctccgt tgggccacct acagataacc 360gcggtctccc ggcagctctt gagcgccatc gactacgtcc actgcgaagg catcatccac 420cgcgatatta 430 <210> SEQ ID NO 10 <211> LENGTH: 22 <212> TYPE: PRT <213>ORGANISM: Herpes simplex virus <400> SEQUENCE: 10 Trp Thr Gly Arg HisPhe Gly Pro Ser Ala Ala Gly Ala Ser Pro Leu 1 5 10 15 Arg Pro Trp GlnAsn Trp 20 <210> SEQ ID NO 11 <211> LENGTH: 143 <212> TYPE: PRT <213>ORGANISM: Herpes simplex virus <400> SEQUENCE: 11 Met Asp Arg Glu AlaLeu Arg Ala Ile Ser Arg Gly Cys Lys Pro Pro 1 5 10 15 Ser Thr Leu AlaLys Leu Val Thr Gly Leu Gly Phe Ala Ile His Gly 20 25 30 Ala Leu Ile ProGly Ser Glu Gly Cys Val Phe Asp Ser Ser His Pro 35 40 45 Asn Tyr Pro HisArg Val Ile Val Lys Ala Gly Trp Tyr Ala Ser Thr 50 55 60 Asn His Glu AlaArg Leu Leu Arg Arg Leu Asn His Pro Ala Ile Leu 65 70 75 80 Pro Leu LeuAsp Leu His Val Val Ser Gly Val Thr Cys Leu Val Leu 85 90 95 Pro Lys TyrHis Cys Asp Leu Tyr Thr Tyr Leu Ser Lys Arg Pro Ser 100 105 110 Pro LeuGly His Leu Gln Ile Thr Ala Val Ser Arg Gln Leu Leu Ser 115 120 125 AlaIle Asp Tyr Val His Cys Glu Gly Ile Ile His Arg Asp Ile 130 135 140<210> SEQ ID NO 12 <211> LENGTH: 481 <212> TYPE: PRT <213> ORGANISM:Herpes simplex virus <400> SEQUENCE: 12 Met Ala Cys Arg Lys Phe Cys GlyVal Tyr Arg Arg Pro Asp Lys Arg 1 5 10 15 Gln Glu Ala Ser Val Pro ProGlu Thr Asn Thr Ala Pro Ala Phe Pro 20 25 30 Ala Ser Thr Phe Tyr Thr ProAla Glu Asp Ala Tyr Leu Ala Pro Gly 35 40 45 Pro Pro Glu Thr Ile His ProSer Arg Pro Pro Ser Pro Gly Glu Ala 50 55 60 Ala Arg Leu Cys Gln Leu GlnGlu Ile Leu Ala Gln Met His Ser Asp 65 70 75 80 Glu Asp Tyr Pro Ile ValAsp Ala Ala Gly Ala Glu Glu Glu Asp Glu 85 90 95 Ala Asp Asp Asp Ala ProAsp Asp Val Ala Tyr Pro Glu Asp Tyr Ala 100 105 110 Glu Gly Arg Phe LeuSer Met Val Ser Ala Ala Pro Leu Pro Gly Ala 115 120 125 Ser Gly His ProPro Val Pro Gly Arg Ala Ala Pro Pro Asp Val Arg 130 135 140 Thr Cys AspThr Gly Lys Val Gly Ala Thr Gly Phe Thr Pro Glu Glu 145 150 155 160 LeuAsp Thr Met Asp Arg Glu Ala Leu Arg Ala Ile Ser Arg Gly Cys 165 170 175Lys Pro Pro Ser Thr Leu Ala Lys Leu Val Thr Gly Leu Gly Phe Ala 180 185190 Ile His Gly Ala Leu Ile Pro Gly Ser Glu Gly Cys Val Phe Asp Ser 195200 205 Ser His Pro Asn Tyr Pro His Arg Val Ile Val Lys Ala Gly Trp Tyr210 215 220 Ala Ser Thr Ser His Glu Ala Arg Leu Leu Arg Arg Leu Asn HisPro 225 230 235 240 Ala Ile Leu Pro Leu Leu Asp Leu His Val Val Ser GlyVal Thr Cys 245 250 255 Leu Val Leu Pro Lys Tyr His Cys Asp Leu Tyr ThrTyr Leu Ser Lys 260 265 270 Arg Pro Ser Pro Leu Gly His Leu Gln Ile ThrAla Val Ser Arg Gln 275 280 285 Leu Leu Ser Ala Ile Asp Tyr Val His CysLys Gly Ile Ile His Arg 290 295 300 Asp Ile Lys Thr Glu Asn Ile Phe IleAsn Thr Pro Glu Asn Ile Cys 305 310 315 320 Leu Gly Asp Phe Gly Ala AlaCys Phe Val Arg Gly Cys Arg Ser Ser 325 330 335 Pro Phe His Tyr Gly IleAla Gly Thr Ile Asp Thr Asn Ala Pro Glu 340 345 350 Val Leu Ala Gly AspPro Tyr Thr Gln Val Ile Asp Ile Trp Ser Ala 355 360 365 Gly Leu Val IlePhe Glu Thr Ala Val His Thr Ala Ser Leu Phe Ser 370 375 380 Ala Pro ArgAsp Pro Glu Arg Arg Pro Cys Asp Asn Gln Ile Ala Arg 385 390 395 400 IleIle Arg Gln Ala Gln Val His Val Asp Glu Phe Pro Thr His Ala 405 410 415Glu Ser Arg Leu Thr Ala His Tyr Arg Ser Arg Ala Ala Gly Asn Asn 420 425430 Arg Pro Ala Trp Thr Arg Pro Ala Trp Thr Arg Tyr Tyr Lys Ile His 435440 445 Thr Asp Val Glu Tyr Leu Ile Cys Lys Ala Leu Thr Phe Asp Ala Ala450 455 460 Leu Arg Pro Ser Ala Ala Glu Leu Leu Arg Leu Pro Leu Phe HisPro 465 470 475 480 Lys <210> SEQ ID NO 13 <211> LENGTH: 501 <212> TYPE:DNA <213> ORGANISM: Herpes simplex virus <400> SEQUENCE: 13 gggggcgcgtctacgaggag atcccctggg ttcgggtata cgaaaacatc tgccttcgcc 60 ggcaagacgccggcggggcg gccccgccgg gagacgcccc ggactccccg tacatcgagg 120 cggaaaatcccctgtacgac tggggcgggt ctgccctctt ctcccctccg ggggccacac 180 gcgccccggacccgggacta agcctgtcgc ccatgcccgc ccgcccccgg accaacgcgc 240 tggccaacgacggcccgaca aacgtcgccg ccctcagcgc cctgttgacg aagctcaaac 300 gcggccgacaccagagccat taaaaaaatg cgaccgccgg ccccaccgtc tcggtttccg 360 gcccctttccccgtatgtct gttttcaata aaaagtaaca aacagagaaa aaaaaacagc 420 gagttccgcatggtttgtcg tacgcaatta gctgtttatt gttttttttt tggggggggg 480 aagagaaaaagaaaaaagga g 501 <210> SEQ ID NO 14 <211> LENGTH: 106 <212> TYPE: PRT<213> ORGANISM: Herpes simplex virus <400> SEQUENCE: 14 Gly Arg Val TyrGlu Glu Ile Pro Trp Val Arg Val Tyr Glu Asn Ile 1 5 10 15 Cys Leu ArgArg Gln Asp Ala Gly Gly Ala Ala Pro Pro Gly Asp Ala 20 25 30 Pro Asp SerPro Tyr Ile Glu Ala Glu Asn Pro Leu Tyr Asp Trp Gly 35 40 45 Gly Ser AlaLeu Phe Ser Pro Pro Gly Ala Thr Arg Ala Pro Asp Pro 50 55 60 Gly Leu SerLeu Ser Pro Met Pro Ala Arg Pro Arg Thr Asn Ala Leu 65 70 75 80 Ala AsnAsp Gly Pro Thr Asn Val Ala Ala Leu Ser Ala Leu Leu Thr 85 90 95 Lys LeuLys Arg Gly Arg His Gln Ser His 100 105 <210> SEQ ID NO 15 <211> LENGTH:722 <212> TYPE: PRT <213> ORGANISM: Herpes simplex virus <400> SEQUENCE:15 Met Gln Arg Arg Ala Arg Gly Ala Ser Ser Leu Arg Leu Ala Arg Cys 1 510 15 Leu Thr Pro Ala Asn Leu Ile Arg Gly Ala Asn Ala Gly Val Pro Glu 2025 30 Arg Arg Ile Phe Ala Gly Cys Leu Leu Pro Thr Pro Glu Gly Leu Leu 3540 45 Ser Ala Ala Val Gly Val Leu Arg Gln Arg Ala Asp Asp Leu Gln Pro 5055 60 Ala Phe Leu Thr Gly Ala Asp Arg Ser Val Arg Leu Ala Ala Arg His 6570 75 80 His Asn Thr Val Pro Glu Ser Leu Ile Val Asp Gly Leu Ala Ser Asp85 90 95 Pro His Tyr Asp Tyr Ile Arg His Tyr Ala Ser Ala Ala Lys Gln Ala100 105 110 Leu Gly Glu Val Glu Leu Ser Gly Gly Gln Leu Ser Arg Ala IleLeu 115 120 125 Ala Gln Tyr Trp Lys Tyr Leu Gln Thr Val Val Pro Ser GlyLeu Asp 130 135 140 Ile Pro Asp Asp Pro Ala Gly Asp Cys Asp Pro Ser LeuHis Val Leu 145 150 155 160 Leu Arg Pro Thr Leu Leu Pro Lys Leu Leu ValArg Ala Pro Phe Lys 165 170 175 Ser Gly Ala Ala Ala Ala Lys Tyr Ala AlaAla Val Ala Gly Leu Arg 180 185 190 Asp Ala Ala His Arg Leu Gln Gln TyrMet Phe Phe Met Arg Pro Ala 195 200 205 Asp Pro Ser Arg Pro Ser Thr AspThr Ala Leu Arg Leu Ser Glu Leu 210 215 220 Leu Ala Tyr Val Ser Val LeuTyr His Trp Ala Ser Trp Met Leu Trp 225 230 235 240 Thr Ala Asp Lys TyrVal Cys Arg Arg Leu Gly Pro Ala Asp Arg Arg 245 250 255 Phe Val Ala LeuSer Gly Ser Leu Glu Ala Pro Ala Glu Thr Phe Ala 260 265 270 Arg His LeuAsp Arg Gly Pro Ser Gly Thr Thr Gly Ser Met Gln Cys 275 280 285 Met AlaLeu Arg Ala Ala Val Ser Asp Val Leu Gly His Leu Thr Arg 290 295 300 LeuAla His Leu Trp Glu Thr Gly Lys Arg Ser Gly Gly Thr Tyr Gly 305 310 315320 Ile Val Asp Ala Ile Val Ser Thr Val Glu Val Leu Ser Ile Val His 325330 335 His His Ala Gln Tyr Ile Ile Asn Ala Thr Leu Thr Gly Tyr Val Val340 345 350 Trp Ala Ser Asp Ser Leu Asn Asn Glu Tyr Leu Thr Ala Ala ValAsp 355 360 365 Ser Gln Glu Arg Phe Cys Arg Thr Ala Ala Pro Leu Phe ProThr Met 370 375 380 Thr Ala Pro Ser Trp Ala Arg Met Glu Leu Ser Ile LysSer Trp Phe 385 390 395 400 Gly Ala Ala Leu Ala Pro Asp Leu Leu Arg SerGly Thr Pro Ser Pro 405 410 415 His Tyr Glu Ser Ile Leu Arg Leu Ala AlaSer Gly Pro Pro Gly Gly 420 425 430 Arg Gly Ala Val Gly Gly Ser Cys ArgAsp Lys Ile Gln Arg Thr Arg 435 440 445 Arg Asp Asn Ala Pro Pro Pro LeuPro Arg Ala Arg Pro His Ser Thr 450 455 460 Pro Ala Ala Pro Arg Arg CysArg Arg His Arg Glu Asp Leu Pro Glu 465 470 475 480 Pro Pro His Val AspAla Ala Asp Arg Gly Pro Glu Pro Cys Ala Gly 485 490 495 Arg Pro Ala ThrTyr Tyr Thr His Met Ala Gly Ala Pro Pro Arg Leu 500 505 510 Pro Pro ArgAsn Pro Ala Pro Pro Glu Gln Arg Pro Ala Ala Ala Ala 515 520 525 Arg ProLeu Ala Ala Gln Arg Glu Ala Ala Gly Val Tyr Asp Ala Val 530 535 540 ArgThr Trp Gly Pro Asp Ala Glu Ala Glu Pro Asp Gln Met Glu Asn 545 550 555560 Thr Tyr Leu Leu Pro Asp Asp Asp Ala Ala Met Pro Ala Gly Val Gly 565570 575 Leu Gly Ala Thr Pro Ala Ala Asp Thr Thr Ala Ala Ala Ala Trp Pro580 585 590 Ala Glu Ser His Ala Pro Arg Ala Pro Ser Glu Asp Ala Asp SerIle 595 600 605 Tyr Glu Ser Val Gly Glu Asp Gly Gly Arg Val Tyr Glu GluIle Pro 610 615 620 Trp Val Arg Val Tyr Glu Asn Ile Cys Pro Arg Arg ArgLeu Ala Gly 625 630 635 640 Gly Ala Ala Leu Pro Gly Asp Ala Pro Asp SerPro Tyr Ile Glu Ala 645 650 655 Glu Asn Pro Leu Tyr Asp Trp Gly Gly SerAla Leu Phe Ser Pro Arg 660 665 670 Arg Ala Thr Arg Ala Pro Asp Pro GlyLeu Ser Leu Ser Pro Met Pro 675 680 685 Ala Arg Pro Arg Thr Asn Ala LeuAla Asn Asp Gly Pro Thr Asn Val 690 695 700 Ala Ala Leu Ser Ala Leu LeuThr Lys Leu Lys Arg Gly Arg His Gln 705 710 715 720 Ser His <210> SEQ IDNO 16 <211> LENGTH: 200 <212> TYPE: DNA <213> ORGANISM: Herpes simplexvirus <400> SEQUENCE: 16 actgcaacgc aatcccatga aggccctgta tccgctcaccaccaaggaac tcaagacttc 60 cgaccccggg ggcgtgggcg gggaggggga ggaaggcgcggaggggggcg ggtttgacga 120 ggccaagttg gccgaggccc gagaaatgat ccgatatatggctttggtgt cggccatgga 180 gcgcacggaa cacaaggcca 200 <210> SEQ ID NO 17<211> LENGTH: 66 <212> TYPE: PRT <213> ORGANISM: Herpes simplex virus<400> SEQUENCE: 17 Leu Gln Arg Asn Pro Met Lys Ala Leu Tyr Pro Leu ThrThr Lys Glu 1 5 10 15 Leu Lys Thr Ser Asp Pro Gly Gly Val Gly Gly GluGly Glu Glu Gly 20 25 30 Ala Glu Gly Gly Gly Phe Asp Glu Ala Lys Leu AlaGlu Ala Arg Glu 35 40 45 Met Ile Arg Tyr Met Ala Leu Val Ser Ala Met GluArg Thr Glu His 50 55 60 Lys Ala 65 <210> SEQ ID NO 18 <211> LENGTH: 904<212> TYPE: PRT <213> ORGANISM: Herpes simplex virus <400> SEQUENCE: 18Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu Val 1 5 1015 Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro Arg Ala 20 2530 Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro Ala Ser 35 4045 Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg 50 5560 Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp 65 7075 80 Ala Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His Leu 8590 95 Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro100 105 110 Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg ArgCys 115 120 125 Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile AlaVal Val 130 135 140 Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala ThrMet Tyr Tyr 145 150 155 160 Lys Asp Val Thr Val Ser Gln Val Trp Phe GlyHis Arg Tyr Ser Gln 165 170 175 Phe Met Gly Ile Phe Glu Asp Arg Ala ProVal Pro Phe Glu Glu Val 180 185 190 Ile Asp Lys Ile Asn Thr Lys Gly ValCys Arg Ser Thr Ala Lys Tyr 195 200 205 Val Arg Asn Asn Met Glu Thr ThrAla Phe His Arg Asp Asp His Glu 210 215 220 Thr Asp Met Glu Leu Lys ProAla Lys Val Ala Thr Arg Thr Ser Arg 225 230 235 240 Gly Trp His Thr ThrAsp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala 245 250 255 Phe His Arg TyrGly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp 260 265 270 Ala Arg SerVal Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp 275 280 285 Phe ValTyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr 290 295 300 GluHis Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe 305 310 315320 Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr 325330 335 Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val340 345 350 Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu ValAsp 355 360 365 Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe SerSer Asp 370 375 380 Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Glu TyrSer Leu Ser 385 390 395 400 Arg Val Asp Leu Gly Asp Cys Ile Gly Arg AspAla Arg Glu Ala Ile 405 410 415 Asp Arg Met Phe Ala Arg Lys Tyr Asn AlaThr His Ile Lys Val Gly 420 425 430 Gln Pro Gln Tyr Tyr Leu Ala Thr GlyGly Phe Leu Ile Ala Tyr Gln 435 440 445 Pro Leu Leu Ser Asn Thr Leu AlaGlu Leu Tyr Val Arg Glu Tyr Met 450 455 460 Arg Glu Gln Asp Arg Lys ProArg Asn Ala Thr Pro Ala Pro Leu Arg 465 470 475 480 Glu Ala Pro Ser AlaAsn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser 485 490 495 Ser Ile Glu PheAla Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Arg 500 505 510 His Val AsnAsp Met Leu Gly Arg Ile Ala Val Ala Trp Cys Glu Leu 515 520 525 Gln AsnHis Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro 530 535 540 AsnAla Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met 545 550 555560 Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp 565570 575 Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr580 585 590 Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln GlyPro 595 600 605 Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg LeuThr Arg 610 615 620 Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg TyrPhe Ile Phe 625 630 635 640 Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr AlaTyr Ser His Gln Leu 645 650 655 Ser Arg Ala Asp Val Thr Thr Val Ser ThrPhe Ile Asp Leu Asn Ile 660 665 670 Thr Met Leu Glu Asp His Glu Phe ValPro Leu Glu Val Tyr Thr Arg 675 680 685 His Glu Ile Lys Asp Ser Gly LeuLeu Asp Tyr Thr Glu Val Gln Arg 690 695 700 Arg Asn Gln Leu His Asp LeuArg Phe Ala Asp Ile Asp Thr Val Ile 705 710 715 720 Arg Ala Asp Ala AsnAla Ala Met Phe Ala Gly Leu Cys Ala Phe Phe 725 730 735 Glu Gly Met GlyAsp Leu Gly Arg Ala Val Gly Lys Val Val Met Gly 740 745 750 Val Val GlyGly Val Val Ser Ala Val Ser Gly Val Ser Ser Phe Met 755 760 765 Ser AsnPro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly 770 775 780 LeuVal Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg 785 790 795800 Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr 805810 815 Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly820 825 830 Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met IleArg 835 840 845 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His LysAla Arg 850 855 860 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val ThrAsn Met Val 865 870 875 880 Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser ProLeu His Asn Glu Asp 885 890 895 Glu Ala Gly Asp Glu Asp Glu Leu 900<210> SEQ ID NO 19 <211> LENGTH: 443 <212> TYPE: DNA <213> ORGANISM:Herpes simplex virus <400> SEQUENCE: 19 ccctctccca cacggtcggt gccccccatctctgtttcat catcgtcccg gttgcgttgc 60 gctttccggc cctcccgcac ccccgcgttccggtgtctcg cggcccggcg ccatgatcac 120 ggattgtttc gaagcagaca tcgcgatcccctcgggtatc tcgcgccccg atgccgcggc 180 gctgcagcgg tgcgagggtc gagtggtctttctgccgacc atccgccgcc agctggcgct 240 cgcggacgtg gcgcacgaat cgttcgtctccggaggagtt agtcccgaca cgttggggtt 300 gttgctggcg taccgcaggc gcttccccgcggtaatcacg cgggtgctgc ccacgcgaat 360 cgtcgcctgc cccgtggacc tggggctcacgcacgccggc accgtcaatc tccgcaacac 420 ctcccccgtc gacctctgca acg 443 <210>SEQ ID NO 20 <211> LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Herpessimplex virus <400> SEQUENCE: 20 Pro Leu Pro His Gly Arg Cys Pro Pro SerLeu Phe His His Arg Pro 1 5 10 15 Gly Cys Val Ala Leu Ser Gly Pro ProAla Pro Pro Arg Ser Gly Val 20 25 30 Ser Arg Pro Gly Ala 35 <210> SEQ IDNO 21 <211> LENGTH: 147 <212> TYPE: PRT <213> ORGANISM: Herpes simplexvirus <400> SEQUENCE: 21 Pro Leu Pro His Gly Arg Cys Pro Pro Ser Leu PheHis His Arg Pro 1 5 10 15 Gly Cys Val Ala Leu Ser Gly Pro Pro Ala ProPro Arg Ser Gly Val 20 25 30 Ser Arg Pro Gly Ala Met Ile Thr Asp Cys PheGlu Ala Asp Ile Ala 35 40 45 Ile Pro Ser Gly Ile Ser Arg Pro Asp Ala AlaAla Leu Gln Arg Cys 50 55 60 Glu Gly Arg Val Val Phe Leu Pro Thr Ile ArgArg Gln Leu Ala Leu 65 70 75 80 Ala Asp Val Ala His Glu Ser Phe Val SerGly Gly Val Ser Pro Asp 85 90 95 Thr Leu Gly Leu Leu Leu Ala Tyr Arg ArgArg Phe Pro Ala Val Ile 100 105 110 Thr Arg Val Leu Pro Thr Arg Ile ValAla Cys Pro Val Asp Leu Gly 115 120 125 Leu Thr His Ala Gly Thr Val AsnLeu Arg Asn Thr Ser Pro Val Asp 130 135 140 Leu Cys Asn 145 <210> SEQ IDNO 22 <211> LENGTH: 110 <212> TYPE: PRT <213> ORGANISM: Herpes simplexvirus <400> SEQUENCE: 22 Met Ile Thr Asp Cys Phe Glu Ala Asp Ile Ala IlePro Ser Gly Ile 1 5 10 15 Ser Arg Pro Asp Ala Ala Ala Leu Gln Arg CysGlu Gly Arg Val Val 20 25 30 Phe Leu Pro Thr Ile Arg Arg Gln Leu Ala LeuAla Asp Val Ala His 35 40 45 Glu Ser Phe Val Ser Gly Gly Val Ser Pro AspThr Leu Gly Leu Leu 50 55 60 Leu Ala Tyr Arg Arg Arg Phe Pro Ala Val IleThr Arg Val Leu Pro 65 70 75 80 Thr Arg Ile Val Ala Cys Pro Val Asp LeuGly Leu Thr His Ala Gly 85 90 95 Thr Val Asn Leu Arg Asn Thr Ser Pro ValAsp Leu Cys Asn 100 105 110 <210> SEQ ID NO 23 <211> LENGTH: 318 <212>TYPE: PRT <213> ORGANISM: Herpes simplex virus <400> SEQUENCE: 23 MetIle Thr Asp Cys Phe Glu Ala Asp Ile Ala Ile Pro Ser Gly Ile 1 5 10 15Ser Arg Pro Asp Ala Ala Ala Leu Gln Arg Cys Glu Gly Arg Val Val 20 25 30Phe Leu Pro Thr Ile Arg Arg Gln Leu Ala Leu Ala Asp Val Ala His 35 40 45Glu Ser Phe Val Ser Gly Gly Val Ser Pro Asp Thr Leu Gly Leu Leu 50 55 60Leu Ala Tyr Arg Arg Arg Phe Pro Ala Val Ile Thr Arg Val Leu Pro 65 70 7580 Thr Arg Ile Val Ala Cys Pro Val Asp Leu Gly Leu Thr His Ala Gly 85 9095 Thr Val Asn Leu Arg Asn Thr Ser Pro Val Asp Leu Cys Asn Gly Asp 100105 110 Pro Val Ser Leu Val Pro Pro Val Phe Glu Gly Gln Ala Thr Asp Val115 120 125 Arg Leu Glu Ser Leu Asp Leu Thr Leu Arg Phe Pro Val Pro LeuPro 130 135 140 Thr Pro Leu Ala Arg Glu Ile Val Ala Arg Leu Val Ala ArgGly Ile 145 150 155 160 Arg Asp Leu Asn Pro Asp Pro Arg Thr Pro Gly GluLeu Pro Asp Leu 165 170 175 Asn Val Leu Tyr Tyr Asn Gly Ala Arg Leu SerLeu Val Ala Asp Val 180 185 190 Gln Gln Leu Ala Ser Val Asn Thr Glu LeuArg Ser Leu Val Leu Asn 195 200 205 Met Val Tyr Ser Ile Thr Glu Gly ThrThr Leu Ile Leu Thr Leu Ile 210 215 220 Pro Arg Leu Leu Ala Leu Ser AlaGln Asp Gly Tyr Val Asn Ala Leu 225 230 235 240 Leu Gln Met Gln Ser ValThr Arg Glu Ala Ala Gln Leu Ile His Pro 245 250 255 Glu Ala Pro Met LeuMet Gln Asp Gly Glu Arg Arg Leu Pro Leu Tyr 260 265 270 Glu Ala Leu ValAla Trp Leu Ala His Ala Gly Gln Leu Gly Asp Ile 275 280 285 Leu Ala LeuAla Pro Ala Val Arg Val Cys Thr Phe Asp Gly Ala Ala 290 295 300 Val ValGln Ser Gly Asp Met Ala Pro Val Ile Arg Tyr Pro 305 310 315 <210> SEQ IDNO 24 <211> LENGTH: 502 <212> TYPE: DNA <213> ORGANISM: Herpes simplexvirus <400> SEQUENCE: 24 actgttgtag gggggaaaac acagttccgg gaaggcgtttattgcggaga gaggggggaa 60 agaaagagaa acaaaagaaa cggcaagaaa gactcaagacgtgcgcgtga tcggaaaaaa 120 ggccgggggg atcccggtcg gggccgccag gtaaatggccatgatgaccg cgaccatgag 180 gtcgtccgcg gcaccgttgc gttttccgga gtacatgcggacgtcggtgt tgggagagac 240 ggtttcgatg aggttgttga gctgctcgga cagatactcgaccgggtcgg tctgcaggcg 300 caccgtcacg gagacgagct cctgggacgc catgacgcccccggagttga actttttgat 360 aaagtattcg aaggcgggcg tcttctgttt gttgagcagaaagaaggggt acaataccgc 420 gccgccgggc ggctcgcagt gatagaagag gagctcgggccccgggccgt tggcccccgc 480 cgaggccagg atgcggtgca tc 502 <210> SEQ ID NO25 <211> LENGTH: 135 <212> TYPE: PRT <213> ORGANISM: Herpes simplexvirus <400> SEQUENCE: 25 Met His Arg Ile Leu Ala Ser Ala Gly Ala Asn GlyPro Gly Pro Glu 1 5 10 15 Leu Leu Phe Tyr His Cys Glu Pro Pro Gly GlyAla Val Leu Tyr Pro 20 25 30 Phe Phe Leu Leu Asn Lys Gln Lys Thr Pro AlaPhe Glu Tyr Phe Ile 35 40 45 Lys Lys Phe Asn Ser Gly Gly Val Met Ala SerGln Glu Leu Val Ser 50 55 60 Val Thr Val Arg Leu Gln Thr Asp Pro Val GluTyr Leu Ser Glu Gln 65 70 75 80 Leu Asn Asn Leu Ile Glu Thr Val Ser ProAsn Thr Asp Val Arg Met 85 90 95 Tyr Ser Gly Lys Arg Asn Gly Ala Ala AspAsp Leu Met Val Ala Val 100 105 110 Ile Met Ala Ile Tyr Leu Ala Ala ProThr Gly Ile Pro Pro Ala Phe 115 120 125 Phe Pro Ile Thr Arg Thr Ser 130135 <210> SEQ ID NO 26 <211> LENGTH: 734 <212> TYPE: PRT <213> ORGANISM:Herpes simplex virus <400> SEQUENCE: 26 Met Phe Gly Gln Gln Leu Ala SerAsp Val Gln Gln Tyr Leu Glu Arg 1 5 10 15 Leu Glu Lys Gln Arg Gln GlnLys Val Gly Val Asp Glu Ala Ser Ala 20 25 30 Gly Leu Thr Leu Gly Gly AspAla Leu Arg Val Pro Phe Leu Asp Phe 35 40 45 Ala Thr Ala Thr Pro Lys ArgHis Gln Thr Val Val Pro Gly Val Gly 50 55 60 Thr Leu His Asp Cys Cys GluHis Ser Pro Leu Phe Ser Ala Val Ala 65 70 75 80 Arg Arg Leu Leu Phe AsnSer Leu Val Pro Ala Gln Leu Arg Gly Arg 85 90 95 Asp Phe Gly Gly Asp HisThr Ala Lys Leu Glu Phe Leu Ala Pro Glu 100 105 110 Leu Val Arg Ala ValAla Arg Leu Arg Phe Arg Glu Cys Ala Pro Glu 115 120 125 Asp Ala Val ProGln Arg Asn Ala Tyr Tyr Ser Val Leu Asn Thr Phe 130 135 140 Gln Ala LeuHis Arg Ser Glu Ala Phe Arg Gln Leu Val His Phe Val 145 150 155 160 ArgAsp Phe Ala Gln Leu Leu Lys Thr Ser Phe Arg Ala Ser Ser Leu 165 170 175Ala Glu Thr Thr Gly Pro Pro Lys Lys Arg Ala Lys Val Asp Val Ala 180 185190 Thr His Gly Gln Thr Tyr Gly Thr Leu Glu Leu Phe Gln Lys Met Ile 195200 205 Leu Met His Ala Thr Tyr Phe Leu Ala Ala Val Leu Leu Gly Asp His210 215 220 Ala Glu Gln Val Asn Thr Phe Leu Arg Leu Val Phe Glu Ile ProLeu 225 230 235 240 Phe Ser Asp Thr Ala Val Arg His Phe Arg Gln Arg AlaThr Val Phe 245 250 255 Leu Val Pro Arg Arg His Gly Lys Thr Trp Phe LeuVal Pro Leu Ile 260 265 270 Ala Leu Ser Leu Ala Ser Phe Arg Gly Ile LysIle Gly Tyr Thr Ala 275 280 285 His Ile Arg Lys Ala Thr Glu Pro Val PheAsp Glu Ile Asp Ala Cys 290 295 300 Leu Arg Gly Trp Phe Gly Ser Ser ArgVal Asp His Val Lys Gly Glu 305 310 315 320 Thr Ile Ser Phe Ser Phe ProAsp Gly Ser Arg Ser Thr Ile Val Phe 325 330 335 Ala Ser Ser His Asn ThrAsn Gly Ile Arg Gly Gln Asp Phe Asn Leu 340 345 350 Leu Phe Val Asp GluAla Asn Phe Ile Arg Pro Asp Ala Val Gln Thr 355 360 365 Ile Met Gly PheLeu Asn Gln Ala Asn Cys Lys Ile Ile Phe Val Ser 370 375 380 Ser Thr AsnThr Gly Lys Ala Ser Thr Ser Phe Leu Tyr Asn Leu Arg 385 390 395 400 GlyAla Ala Asp Glu Leu Leu Asn Val Val Thr Tyr Ile Cys Asp Asp 405 410 415His Met Pro Arg Val Val Thr His Thr Asn Ala Thr Ala Cys Ser Cys 420 425430 Tyr Ile Leu Asn Lys Pro Val Phe Ile Thr Met Asp Gly Ala Val Arg 435440 445 Arg Thr Ala Asp Leu Phe Leu Pro Asp Ser Phe Met Gln Glu Ile Ile450 455 460 Gly Gly Gln Ala Arg Glu Thr Gly Asp Asp Arg Pro Val Leu ThrLys 465 470 475 480 Ser Ala Gly Glu Arg Phe Leu Leu Tyr Arg Pro Ser ThrThr Thr Asn 485 490 495 Ser Gly Leu Met Ala Pro Glu Leu Tyr Val Tyr ValAsp Pro Ala Phe 500 505 510 Thr Ala Asn Thr Arg Ala Ser Gly Thr Gly IleAla Val Val Gly Arg 515 520 525 Tyr Arg Asp Asp Phe Ile Ile Phe Ala LeuGlu His Phe Phe Leu Arg 530 535 540 Ala Leu Thr Gly Ser Ala Pro Ala AspIle Ala Arg Cys Val Val His 545 550 555 560 Ser Leu Ala Gln Val Leu AlaLeu His Pro Gly Ala Phe Arg Ser Val 565 570 575 Arg Val Ala Val Glu GlyAsn Ser Ser Gln Asp Ser Ala Val Ala Ile 580 585 590 Ala Thr His Val HisThr Glu Met His Arg Ile Leu Ala Ser Ala Gly 595 600 605 Ala Asn Gly ProGly Pro Glu Leu Leu Phe Tyr His Cys Glu Pro Pro 610 615 620 Gly Gly AlaVal Leu Tyr Pro Phe Phe Leu Leu Asn Lys Gln Lys Thr 625 630 635 640 ProAla Phe Glu Tyr Phe Ile Lys Lys Phe Asn Ser Gly Gly Val Met 645 650 655Ala Ser Gln Glu Leu Val Ser Val Thr Val Arg Leu Gln Thr Asp Pro 660 665670 Val Glu Tyr Leu Ser Glu Gln Leu Asn Asn Leu Ile Glu Thr Val Ser 675680 685 Pro Asn Thr Asp Val Arg Met Tyr Ser Gly Lys Arg Asn Gly Ala Ala690 695 700 Asp Asp Leu Met Val Ala Val Ile Met Ala Ile Tyr Leu Ala AlaPro 705 710 715 720 Thr Gly Ile Pro Pro Ala Phe Phe Pro Ile Thr Arg ThrSer 725 730 <210> SEQ ID NO 27 <211> LENGTH: 15 <212> TYPE: PRT <213>ORGANISM: HSV-2 <400> SEQUENCE: 27 Gly Arg Val Tyr Glu Glu Ile Pro TrpVal Arg Val Tyr Glu Asn 5 10 15 <210> SEQ ID NO 28 <211> LENGTH: 15<212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 28 Tyr Glu Asn IleCys Leu Arg Arg Gln Asp Ala Gly Gly Ala Ala 5 10 15 <210> SEQ ID NO 29<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE:29 Pro Asp Ser Pro Tyr Ile Glu Ala Glu Asn Pro Leu Tyr Asp Trp 5 10 15<210> SEQ ID NO 30 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 30 Tyr Ile Glu Ala Glu Asn Pro Leu Tyr Asp Trp GlyGly Ser Ala 5 10 15 <210> SEQ ID NO 31 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: HSV-2 <400> SEQUENCE: 31 Thr Asn Ala Leu Ala Asn Asp GlyPro Thr Asn Val Ala Ala Leu 5 10 15 <210> SEQ ID NO 32 <211> LENGTH: 15<212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 32 Arg Val Leu ProThr Arg Ile Val Ala Cys Pro Val Asp Leu Gly 5 10 15 <210> SEQ ID NO 33<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE:33 Thr Arg Ile Val Ala Cys Pro Val Asp Leu Gly Leu Thr His Ala 5 10 15<210> SEQ ID NO 34 <211> LENGTH: 661 <212> TYPE: DNA <213> ORGANISM:HSV-2 <400> SEQUENCE: 34 ctcctcttcc gcctcctcct cctcctcttc cgcctcctcctcctcctcct ccgcctcttc 60 ctctgcgggc ggggctggtg ggagcgtcgc gtccgcgtccggcgctgggg agagacgaga 120 aacctccctc ggcccccgcg ctgctgcgcc gcgggggccgaggaagtgtg ccaggaagac 180 gcgccacgcg gagggcggcc ccgagcccgg ggcccgcgacccggcgcccg gcctcacgcg 240 ctacctgccc atcgcggggg tctcgagcgt cgtggccctggcgccttacg tgaacaagac 300 ggtcacgggg gactgcctgc ccgtcctgga catggagacgggccacatag gggcctacgt 360 ggtcctcgtg gaccagacgg ggaacgtggc ggacctgctgcgggccgcgg cccccgcgtg 420 gagccgccgc accctgctcc ccgagcacgc gcgcaactgcgtgaggcccc ccgactaccc 480 gacgcccccc gcgtcggagt ggaacagcct ctggatgaccccggtgggca acatgctctt 540 tgaccagggc accctggtgg gcgcgctgga cttccacggcctccggtcgc gccacccgtg 600 gtctcgggag cagggcgcgc ccgcgccggc cggcgacgcccccgcgggcc acggggagta 660 g 661 <210> SEQ ID NO 35 <211> LENGTH: 2481<212> TYPE: DNA <213> ORGANISM: HSV-2 <400> SEQUENCE: 35 atggaaccccggcccggcac gagctcccgg gcggaccccg gccccgagcg gccgccgcgg 60 cagacccccggcacgcagcc cgccgccccg cacgcctggg ggatgctcaa cgacatgcag 120 tggctcgccagcagcgactc ggaggaggag accgaggtgg gaatctctga cgacgacctt 180 caccgcgactccacctccga ggcgggcagc acggacacgg agatgttcga ggcgggcctg 240 atggacgcggccacgccccc ggcccggccc ccggccgagc gccagggcag ccccacgccc 300 gccgacgcgcagggatcctg tgggggtggg cccgtgggtg aggaggaagc ggaagcggga 360 ggggggggcgacgtgtgtgc cgtgtgcacg gacgagatcg ccccgcccct gcgctgccag 420 agttttccctgcctgcaccc cttctgcatc ccgtgcatga agacctggat tccgttgcgc 480 aacacgtgtcccctgtgcaa caccccggtg gcgtacctga tagtgggcgt gaccgccagc 540 gggtcgttcagcaccatccc gatagtgaac gacccccgga cccgcgtgga ggccgaggcg 600 gccgtgcgggccggcacggc cgtggacttt atctggacgg gcaacccgcg gacggccccg 660 cgctccctgtcgctgggggg acacacggtc cgcgccctgt cgcccacccc cccgtggccc 720 ggcacggacgacgaggacga tgacctggcc gacggtgtgg actacgtccc gcccgccccc 780 cgaagagcgccccggcgcgg gggcggcggt gcgggggcga cccgcggaac ctcccagccc 840 gccgcgacccgaccggcgcc ccctggcgcc ccgcggagca gcagcagcgg cggcgccccg 900 ttgcgggcgggggtgggatc tgggtctggg ggcggccctg ccgtcgcggc cgtcgtgccg 960 agagtggcctctcttccccc tgcggccggc ggggggcgcg cgcaggcgcg gcgggtgggc 1020 gaagacgccgcggcggcgga gggcaggacg ccccccgcga gacagccccg cgcggcccag 1080 gagccccccatagtcatcag cgactctccc ccgccgtctc cgcgccgccc cgcgggcccc 1140 gggccgctctcctttgtctc ctcctcctcc gcacaggtgt cctcgggccc cgggggggga 1200 ggtctgccacagtcgtcggg gcgcgccgcg cgcccccgcg cggccgtcgc cccgcgcgtc 1260 cggagtccgccccgcgccgc cgccgccccc gtggtgtctg cgagcgcgga cgcggccggg 1320 cccgcgccgcccgccgtgcc ggtggacgcg caccgcgcgc cccggtcgcg catgacccag 1380 gctcagaccgacacccaagc acagagtctg ggccgggcag gcgcgaccga cgcgcgcggg 1440 tcgggagggccgggcgcgga gggaggaccc ggggtccccc gcggcaccaa cacccccggt 1500 gccgccccccacgccgcgga gggggcggcg gcccgccccc ggaagaggcg cgggtcggac 1560 tcgggccccgcggcctcgtc ctccgcctct tcctccgccg ccccgcgctc gcccctcgcc 1620 ccccagggggtgggggccaa gagggcggcg ccgcgccggg ccccggactc ggactcgggc 1680 gaccgcggccacgggccgct cgccccggcg tccgcgggcg ccgcgccccc gtcggcgtct 1740 ccgtcgtcccaggccgcggt cgccgccgcc tcctcctcct ccgcctcctc ctcctccgcc 1800 tcctcctcctccgcctcctc ctcctccgcc tcctcctcct ccgcctcctc ctcctccgcc 1860 tcctcctcctccgcctcttc ctctgcgggc ggggctggtg ggagcgtcgc gtccgcgtcc 1920 ggcgctggggagagacgaga aacctccctc ggcccccgcg ctgctgcgcc gcgggggccg 1980 aggaagtgtgccaggaagac gcgccacgcg gagggcggcc ccgagcccgg ggcccgcgac 2040 ccggcgcccggcctcacgcg ctacctgccc atcgcggggg tctcgagcgt cgtggccctg 2100 gcgccttacgtgaacaagac ggtcacgggg gactgcctgc ccgtcctgga catggagacg 2160 ggccacataggggcctacgt ggtcctcgtg gaccagacgg ggaacgtggc ggacctgctg 2220 cgggccgcggcccccgcgtg gagccgccgc accctgctcc ccgagcacgc gcgcaactgc 2280 gtgaggccccccgactaccc gacgcccccc gcgtcggagt ggaacagcct ctggatgacc 2340 ccggtgggcaacatgctctt tgaccagggc accctggtgg gcgcgctgga cttccacggc 2400 ctccggtcgcgccacccgtg gtctcgggag cagggcgcgc ccgcgccggc cggcgacgcc 2460 cccgcgggccacggggagta g 2481 <210> SEQ ID NO 36 <211> LENGTH: 1603 <212> TYPE: DNA<213> ORGANISM: HSV-2 <400> SEQUENCE: 36 cggccggagg gctgtcccgcatcgatatca cgagccccat gaagcccttc ccgtatcgcg 60 cgcgcacgag cgcggcgtcgcacccgaacg ccagcccgcc cgtcgtccag acgcccacgg 120 gccacgtcga ggccgacggggagaggtaca cgtaccgacc cggagtccgt agcaggcccc 180 tggcggccag ccaggtcacggatgcgttgt gcagatgcgc gatgctcagg ttcgtcgtcg 240 gatgcctcgg tgtccccgcgggcggccccg ggggcggcgc gttgcgtcgg ccgtccgggt 300 gcctctcggt cgccccgtcgtctccccgcg ggaacgtaag cccctcgcgg tccgcgcggc 360 cgcgaatgtt acccaggcccgggaccgcaa cagcgcggag gcgccggggt tgtgcgacag 420 tcccttgagc tgggtcacctcggcgggggg acgggacgtg ggccccgcct cggggagctc 480 gggcaggctc gcgttccgaggccggccgag cagataggtc tttgggatgt aaagcagctg 540 cccggggtcc cgaggaaactcggccgtggt gaccaacacg aaacaaaagc gctcggcgta 600 ccaccgaagc atgggcacggatgccgtagt caggttgagt tcgcccgggg gcgccaagcg 660 tccgcgctgg gggtcgctggcgtcgggggt tgttgggcaa ccacagacgc ccggtgtttt 720 gtcgcgccag tacgtgcgggccaaccccag accgtgcaaa aaccacgggt cgatttgctc 780 cgtccagtac gtgtcatggcccccggcaac gcccaccagg acccccatca ccacccacag 840 accggggccc atggtcgtccgtcccggctg ccagtccgca gatggggggg ggtgtccgta 900 cccacggccc aaagaggctccgcacctcgg aggctatcgg aggccctttg ttgccgtaag 960 cgcgggccaa aggatggggtggggtgaggg taaaagcaca aagggagtac cagaccgaaa 1020 acaaggacgg atcggcccgctccgtttttc ggtggggtgc tgatacggtg ccagccctgg 1080 ccccgaaccc ccgcgcttatggacacacca cacgacaaca atgcctttta ttctgttctt 1140 ttattgccgt catcgccgggaggccttccg ttcgggcttc cgtgtttgaa ctaaactccc 1200 cccacctcgc gggcaaacgtgcgcgccagg tcgcgtatct cggcgatgga cccggcggtt 1260 gtgacgcggg ttgggatcatcccggcggtg aggcgcaaca gggcgtctcg acacccgacg 1320 ggcgactgat cgtaatccaggacaaataga tgcatcggaa ggaggcggtc ggccaagacg 1380 tccaagaccc aggcaaaaatgtggtacaag tccccgttgg gggccagcag ctcgggaacg 1440 cggaacaggg caaacagcgtgtcctcgatg cggggcagag accccgcgcc gtcctcgggg 1500 tcggggcgcg gggtcgccgcggcgaccccc gtcagccggc cccagtcctc ccgccacctc 1560 ccgccgcgct gcaggtaccgcaccgtgttg gcgagtagat cgt 1603 <210> SEQ ID NO 37 <211> LENGTH: 1131<212> TYPE: DNA <213> ORGANISM: HSV-2 <400> SEQUENCE: 37 atggcttctcacgccggcca acagcacgcg cctgcgttcg gtcaggctgc tcgtgcgagc 60 gggcctaccgacggccgcgc ggcgtcccgt cctagccatc gccagggggc ctccggagcc 120 cgcggggatccggagctgcc cacgctgctg cgggtttata tagacggacc ccacggggtg 180 gggaagaccaccacctccgc gcagctgatg gaggccctgg ggccgcgcga caatatcgtc 240 tacgtccccgagccgatgac ttactggcag gtgctggggg cctccgagac cctgacgaac 300 atctacaacacgcagcaccg tctggaccgc ggcgagatat cggccgggga ggcggcggtg 360 gtaatgaccagcgcccagat aacaatgagc acgccttatg cggcgacgga cgccgttttg 420 gctcctcatatcggggggga ggctgtgggc ccgcaagccc cgcccccggc cctcaccctt 480 gttttcgaccggcaccctat cgcctccctg ctgtgctacc cggccgcgcg gtacctcatg 540 ggaagcatgaccccccaggc cgtgttggcg ttcgtggccc tcatgccccc gaccgcgccc 600 ggcacgaacctggtcctggg tgtccttccg gaggccgaac acgccgaccg cctggccaga 660 cgccaacgcccgggcgagcg gcttgacctg gccatgctgt ccgccattcg ccgtgtctac 720 gatctactcgccaacacggt gcggtacctg cagcgcggcg ggaggtggcg ggaggactgg 780 ggccggctgacgggggtcgc cgcggcgacc ccgcgccccg accccgagga cggcgcgggg 840 tctctgccccgcatcgagga cacgctgttt gccctgttcc gcgttcccga gctgctggcc 900 cccaacggggacttgtacca catttttgcc tgggtcttgg acgtcttggc cgaccgcctc 960 cttccgatgcatctatttgt cctggattac gatcagtcgc ccgtcgggtg tcgagacgcc 1020 ctgttgcgcctcaccgccgg gatgatccca acccgcgtca caaccgccgg gtccatcgcc 1080 gagatacgcgacctggcgcg cacgtttgcc cgcgaggtgg ggggagttta g 1131 <210> SEQ ID NO 38<211> LENGTH: 2517 <212> TYPE: DNA <213> ORGANISM: HSV-2 <400> SEQUENCE:38 atgggccccg gtctgtgggt ggtgatgggg gtcctggtgg gcgttgccgg gggccatgac 60acgtactgga cggagcaaat cgacccgtgg tttttgcacg gtctggggtt ggcccgcacg 120tactggcgcg acacaaacac cgggcgtctg tggttgccca acacccccga cgccagcgac 180ccccagcgcg gacgcttggc gcccccgggc gaactcaacc tgactacggc atccgtgccc 240atgcttcggt ggtacgccga gcgcttttgt ttcgtgttgg tcaccacggc cgagtttcct 300cgggaccccg ggcagctgct ttacatccca aagacctatc tgctcggccg gcctcggaac 360gcgagcctgc ccgagctccc cgaggcgggg cccacgtccc gtccccccgc cgaggtgacc 420cagctcaagg gactgtcgca caaccccggc gcctccgcgc tgttgcggtc ccgggcctgg 480gtaacattcg cggccgcgcc ggaccgcgag gggcttacgt tcccgcgggg agacgacggg 540gcgaccgaga ggcacccgga cggccgacgc aacgcgccgc ccccggggcc gcccgcgggg 600acaccgaggc atccgacgac gaacctgagc atcgcgcatc tgcacaacgc atccgtgacc 660tggctggccg ccaggggcct gctacggact ccgggtcggt acgtgtacct ctccccgtcg 720gcctcgacgt ggcccgtggg cgtctggacg acgggcgggc tggcgttcgg gtgcgacgcc 780gcgctcgtgc gcgcgcgata cgggaagggc ttcatggggc tcgtgatatc gatgcgggac 840agccctccgg ccgagatcat agtggtgcct gcggacaaga ccctcgctcg ggtcggaaat 900ccgaccgacg aaaacgcccc cgcggtgctc cccgggcctc cggccggccc caggtatcgc 960gtctttgtcc tgggggcccc gacgcccgcc gacaacggct cggcgctgga cgccctccgg 1020cgggtggccg gctaccccga ggagagcacg aactacgccc agtatatgtc gcgggcctat 1080gcggagtttt tgggggagga cccgggctcc ggcacggacg cgcgtccgtc cctgttctgg 1140cgcctcgcgg ggctgctcgc ctcgtcgggg tttgcgttcg tcaacgcggc ccacgcccac 1200gacgcgattc gcctctccga cctgctgggc tttttggccc actcgcgcgt gctggccggc 1260ctggccgccc ggggagcagc gggctgcgcg gccgactcgg tgttcctgaa cgtgtccgtg 1320ttggacccgg cggcccgcct gcggctggag gcgcgcctcg ggcatctggt ggccgcgatc 1380ctcgagcgag agcagagcct ggtggcgcac gcgctgggct atcagctggc gttcgtgttg 1440gacagccccg cggcctatgg cgcggtggcc ccgagcgcgg cccgcctgat cgacgccctg 1500tacgccgagt ttctcggcgg ccgcgcgcta accgccccga tggtccgccg agcgctgttt 1560tacgccacgg ccgtcctccg ggcgccgttc ctggcgggcg cgccctcggc cgagcagcgg 1620gaacgcgccc gccggggcct cctcataacc acggccctgt gtacgtccga cgtcgccgcg 1680gcgacccacg ccgatctccg ggccgcgcta gccaggaccg accaccagaa aaacctcttc 1740tggctcccgg accacttttc cccatgcgca gcttccctgc gcttcgatct cgccgagggc 1800gggttcatcc tggacgcgct ggccatggcc acccgatccg acatcccggc ggacgtcatg 1860gcacaacaga cccgcggcgt ggcctccgtt ctcacgcgct gggcgcacta caacgccctg 1920atccgcgcct tcgtcccgga ggccacccac cagtgtagcg gcccgtcgca caacgcggag 1980ccccggatcc tcgtgcccat cacccacaac gccagctacg tcgtcaccca cacccccttg 2040ccccgcggga tcggatacaa gcttacgggc gttgacgtcc gccgcccgct gtttatcacc 2100tatctcaccg ccacctgcga agggcacgcg cgggagattg agccgaagcg gctggtgcgc 2160accgaaaacc ggcgcgacct cggcctcgtg ggggccgtgt ttctgcgcta caccccggcc 2220ggggaggtca tgtcggtgct gctggtggac acggatgcca cccaacagca gctggcccag 2280gggccggtgg cgggcacccc gaacgtgttt tccagcgacg tgccgtccgt ggccctgttg 2340ttgttcccca acggaactgt gattcatctg ctggcctttg acacgctgcc catcgccacc 2400atcgcccccg ggtttctggc cgcgtccgcg ctgggggtcg ttatgattac cgcggccctg 2460gcgggcatcc ttagggtggt ccgaacgtgc gtcccatttt tgtggagacg cgaataa 2517<210> SEQ ID NO 39 <211> LENGTH: 376 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 39 Met Ala Ser His Ala Gly Gln Gln His Ala Pro AlaPhe Gly Gln Ala 5 10 15 Ala Arg Ala Ser Gly Pro Thr Asp Gly Arg Ala AlaSer Arg Pro Ser 20 25 30 His Arg Gln Gly Ala Ser Gly Ala Arg Gly Asp ProGlu Leu Pro Thr 35 40 45 Leu Leu Arg Val Tyr Ile Asp Gly Pro His Gly ValGly Lys Thr Thr 50 55 60 Thr Ser Ala Gln Leu Met Glu Ala Leu Gly Pro ArgAsp Asn Ile Val 65 70 75 80 Tyr Val Pro Glu Pro Met Thr Tyr Trp Gln ValLeu Gly Ala Ser Glu 85 90 95 Thr Leu Thr Asn Ile Tyr Asn Thr Gln His ArgLeu Asp Arg Gly Glu 100 105 110 Ile Ser Ala Gly Glu Ala Ala Val Val MetThr Ser Ala Gln Ile Thr 115 120 125 Met Ser Thr Pro Tyr Ala Ala Thr AspAla Val Leu Ala Pro His Ile 130 135 140 Gly Gly Glu Ala Val Gly Pro GlnAla Pro Pro Pro Ala Leu Thr Leu 145 150 155 160 Val Phe Asp Arg His ProIle Ala Ser Leu Leu Cys Tyr Pro Ala Ala 165 170 175 Arg Tyr Leu Met GlySer Met Thr Pro Gln Ala Val Leu Ala Phe Val 180 185 190 Ala Leu Met ProPro Thr Ala Pro Gly Thr Asn Leu Val Leu Gly Val 195 200 205 Leu Pro GluAla Glu His Ala Asp Arg Leu Ala Arg Arg Gln Arg Pro 210 215 220 Gly GluArg Leu Asp Leu Ala Met Leu Ser Ala Ile Arg Arg Val Tyr 225 230 235 240Asp Leu Leu Ala Asn Thr Val Arg Tyr Leu Gln Arg Gly Gly Arg Trp 245 250255 Arg Glu Asp Trp Gly Arg Leu Thr Gly Val Ala Ala Ala Thr Pro Arg 260265 270 Pro Asp Pro Glu Asp Gly Ala Gly Ser Leu Pro Arg Ile Glu Asp Thr275 280 285 Leu Phe Ala Leu Phe Arg Val Pro Glu Leu Leu Ala Pro Asn GlyAsp 290 295 300 Leu Tyr His Ile Phe Ala Trp Val Leu Asp Val Leu Ala AspArg Leu 305 310 315 320 Leu Pro Met His Leu Phe Val Leu Asp Tyr Asp GlnSer Pro Val Gly 325 330 335 Cys Arg Asp Ala Leu Leu Arg Leu Thr Ala GlyMet Ile Pro Thr Arg 340 345 350 Val Thr Thr Ala Gly Ser Ile Ala Glu IleArg Asp Leu Ala Arg Thr 355 360 365 Phe Ala Arg Glu Val Gly Gly Val 370375 <210> SEQ ID NO 40 <211> LENGTH: 136 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 40 Asp Leu Leu Ala Asn Thr Val Arg Tyr Leu Gln ArgGly Gly Arg Trp 5 10 15 Arg Glu Asp Trp Gly Arg Leu Thr Gly Val Ala AlaAla Thr Pro Arg 20 25 30 Pro Asp Pro Glu Asp Gly Ala Gly Ser Leu Pro ArgIle Glu Asp Thr 35 40 45 Leu Phe Ala Leu Phe Arg Val Pro Glu Leu Leu AlaPro Asn Gly Asp 50 55 60 Leu Tyr His Ile Phe Ala Trp Val Leu Asp Val LeuAla Asp Arg Leu 65 70 75 80 Leu Pro Met His Leu Phe Val Leu Asp Tyr AspGln Ser Pro Val Gly 85 90 95 Cys Arg Asp Ala Leu Leu Arg Leu Thr Ala GlyMet Ile Pro Thr Arg 100 105 110 Val Thr Thr Ala Gly Ser Ile Ala Glu IleArg Asp Leu Ala Arg Thr 115 120 125 Phe Ala Arg Glu Val Gly Gly Val 130135 <210> SEQ ID NO 41 <211> LENGTH: 284 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 41 Met Gly Pro Gly Leu Trp Val Val Met Gly Val LeuVal Gly Val Ala 5 10 15 Gly Gly His Asp Thr Tyr Trp Thr Glu Gln Ile AspPro Trp Phe Leu 20 25 30 His Gly Leu Gly Leu Ala Arg Thr Tyr Trp Arg AspThr Asn Thr Gly 35 40 45 Arg Leu Trp Leu Pro Asn Thr Pro Asp Ala Ser AspPro Gln Arg Gly 50 55 60 Arg Leu Ala Pro Pro Gly Glu Leu Asn Leu Thr ThrAla Ser Val Pro 65 70 75 80 Met Leu Arg Trp Tyr Ala Glu Arg Phe Cys PheVal Leu Val Thr Thr 85 90 95 Ala Glu Phe Pro Arg Asp Pro Gly Gln Leu LeuTyr Ile Pro Lys Thr 100 105 110 Tyr Leu Leu Gly Arg Pro Arg Asn Ala SerLeu Pro Glu Leu Pro Glu 115 120 125 Ala Gly Pro Thr Ser Arg Pro Pro AlaGlu Val Thr Gln Leu Lys Gly 130 135 140 Leu Ser His Asn Pro Gly Ala SerAla Leu Leu Arg Ser Arg Ala Trp 145 150 155 160 Val Thr Phe Ala Ala AlaPro Asp Arg Glu Gly Leu Thr Phe Pro Arg 165 170 175 Gly Asp Asp Gly AlaThr Glu Arg His Pro Asp Gly Arg Arg Asn Ala 180 185 190 Pro Pro Pro GlyPro Pro Ala Gly Thr Pro Arg His Pro Thr Thr Asn 195 200 205 Leu Ser IleAla His Leu His Asn Ala Ser Val Thr Trp Leu Ala Ala 210 215 220 Arg GlyLeu Leu Arg Thr Pro Gly Arg Tyr Val Tyr Leu Ser Pro Ser 225 230 235 240Ala Ser Thr Trp Pro Val Gly Val Trp Thr Thr Gly Gly Leu Ala Phe 245 250255 Gly Cys Asp Ala Ala Leu Val Arg Ala Arg Tyr Gly Lys Gly Phe Met 260265 270 Gly Leu Val Ile Ser Met Arg Asp Ser Pro Pro Ala 275 280 <210>SEQ ID NO 42 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HSV-2<400> SEQUENCE: 42 Ser Leu Pro Arg Ile Glu Asp Thr Leu Phe Ala Leu PheArg Val 5 10 15 <210> SEQ ID NO 43 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: HSV-2 <400> SEQUENCE: 43 Gly Ser Ile Ala Glu Ile Arg AspLeu Ala Arg Thr Phe Ala Arg 5 10 15 <210> SEQ ID NO 44 <211> LENGTH: 16<212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 44 Glu Ile Arg AspLeu Ala Arg Thr Phe Ala Arg Glu Val Gly Gly Val 5 10 15 <210> SEQ ID NO45 <211> LENGTH: 838 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400>SEQUENCE: 45 Met Gly Pro Gly Leu Trp Val Val Met Gly Val Leu Val Gly ValAla 5 10 15 Gly Gly His Asp Thr Tyr Trp Thr Glu Gln Ile Asp Pro Trp PheLeu 20 25 30 His Gly Leu Gly Leu Ala Arg Thr Tyr Trp Arg Asp Thr Asn ThrGly 35 40 45 Arg Leu Trp Leu Pro Asn Thr Pro Asp Ala Ser Asp Pro Gln ArgGly 50 55 60 Arg Leu Ala Pro Pro Gly Glu Leu Asn Leu Thr Thr Ala Ser ValPro 65 70 75 80 Met Leu Arg Trp Tyr Ala Glu Arg Phe Cys Phe Val Leu ValThr Thr 85 90 95 Ala Glu Phe Pro Arg Asp Pro Gly Gln Leu Leu Tyr Ile ProLys Thr 100 105 110 Tyr Leu Leu Gly Arg Pro Arg Asn Ala Ser Leu Pro GluLeu Pro Glu 115 120 125 Ala Gly Pro Thr Ser Arg Pro Pro Ala Glu Val ThrGln Leu Lys Gly 130 135 140 Leu Ser His Asn Pro Gly Ala Ser Ala Leu LeuArg Ser Arg Ala Trp 145 150 155 160 Val Thr Phe Ala Ala Ala Pro Asp ArgGlu Gly Leu Thr Phe Pro Arg 165 170 175 Gly Asp Asp Gly Ala Thr Glu ArgHis Pro Asp Gly Arg Arg Asn Ala 180 185 190 Pro Pro Pro Gly Pro Pro AlaGly Thr Pro Arg His Pro Thr Thr Asn 195 200 205 Leu Ser Ile Ala His LeuHis Asn Ala Ser Val Thr Trp Leu Ala Ala 210 215 220 Arg Gly Leu Leu ArgThr Pro Gly Arg Tyr Val Tyr Leu Ser Pro Ser 225 230 235 240 Ala Ser ThrTrp Pro Val Gly Val Trp Thr Thr Gly Gly Leu Ala Phe 245 250 255 Gly CysAsp Ala Ala Leu Val Arg Ala Arg Tyr Gly Lys Gly Phe Met 260 265 270 GlyLeu Val Ile Ser Met Arg Asp Ser Pro Pro Ala Glu Ile Ile Val 275 280 285Val Pro Ala Asp Lys Thr Leu Ala Arg Val Gly Asn Pro Thr Asp Glu 290 295300 Asn Ala Pro Ala Val Leu Pro Gly Pro Pro Ala Gly Pro Arg Tyr Arg 305310 315 320 Val Phe Val Leu Gly Ala Pro Thr Pro Ala Asp Asn Gly Ser AlaLeu 325 330 335 Asp Ala Leu Arg Arg Val Ala Gly Tyr Pro Glu Glu Ser ThrAsn Tyr 340 345 350 Ala Gln Tyr Met Ser Arg Ala Tyr Ala Glu Phe Leu GlyGlu Asp Pro 355 360 365 Gly Ser Gly Thr Asp Ala Arg Pro Ser Leu Phe TrpArg Leu Ala Gly 370 375 380 Leu Leu Ala Ser Ser Gly Phe Ala Phe Val AsnAla Ala His Ala His 385 390 395 400 Asp Ala Ile Arg Leu Ser Asp Leu LeuGly Phe Leu Ala His Ser Arg 405 410 415 Val Leu Ala Gly Leu Ala Ala ArgGly Ala Ala Gly Cys Ala Ala Asp 420 425 430 Ser Val Phe Leu Asn Val SerVal Leu Asp Pro Ala Ala Arg Leu Arg 435 440 445 Leu Glu Ala Arg Leu GlyHis Leu Val Ala Ala Ile Leu Glu Arg Glu 450 455 460 Gln Ser Leu Val AlaHis Ala Leu Gly Tyr Gln Leu Ala Phe Val Leu 465 470 475 480 Asp Ser ProAla Ala Tyr Gly Ala Val Ala Pro Ser Ala Ala Arg Leu 485 490 495 Ile AspAla Leu Tyr Ala Glu Phe Leu Gly Gly Arg Ala Leu Thr Ala 500 505 510 ProMet Val Arg Arg Ala Leu Phe Tyr Ala Thr Ala Val Leu Arg Ala 515 520 525Pro Phe Leu Ala Gly Ala Pro Ser Ala Glu Gln Arg Glu Arg Ala Arg 530 535540 Arg Gly Leu Leu Ile Thr Thr Ala Leu Cys Thr Ser Asp Val Ala Ala 545550 555 560 Ala Thr His Ala Asp Leu Arg Ala Ala Leu Ala Arg Thr Asp HisGln 565 570 575 Lys Asn Leu Phe Trp Leu Pro Asp His Phe Ser Pro Cys AlaAla Ser 580 585 590 Leu Arg Phe Asp Leu Ala Glu Gly Gly Phe Ile Leu AspAla Leu Ala 595 600 605 Met Ala Thr Arg Ser Asp Ile Pro Ala Asp Val MetAla Gln Gln Thr 610 615 620 Arg Gly Val Ala Ser Val Leu Thr Arg Trp AlaHis Tyr Asn Ala Leu 625 630 635 640 Ile Arg Ala Phe Val Pro Glu Ala ThrHis Gln Cys Ser Gly Pro Ser 645 650 655 His Asn Ala Glu Pro Arg Ile LeuVal Pro Ile Thr His Asn Ala Ser 660 665 670 Tyr Val Val Thr His Thr ProLeu Pro Arg Gly Ile Gly Tyr Lys Leu 675 680 685 Thr Gly Val Asp Val ArgArg Pro Leu Phe Ile Thr Tyr Leu Thr Ala 690 695 700 Thr Cys Glu Gly HisAla Arg Glu Ile Glu Pro Lys Arg Leu Val Arg 705 710 715 720 Thr Glu AsnArg Arg Asp Leu Gly Leu Val Gly Ala Val Phe Leu Arg 725 730 735 Tyr ThrPro Ala Gly Glu Val Met Ser Val Leu Leu Val Asp Thr Asp 740 745 750 AlaThr Gln Gln Gln Leu Ala Gln Gly Pro Val Ala Gly Thr Pro Asn 755 760 765Val Phe Ser Ser Asp Val Pro Ser Val Ala Leu Leu Leu Phe Pro Asn 770 775780 Gly Thr Val Ile His Leu Leu Ala Phe Asp Thr Leu Pro Ile Ala Thr 785790 795 800 Ile Ala Pro Gly Phe Leu Ala Ala Ser Ala Leu Gly Val Val MetIle 805 810 815 Thr Ala Ala Leu Ala Gly Ile Leu Arg Val Val Arg Thr CysVal Pro 820 825 830 Phe Leu Trp Arg Arg Glu 835 <210> SEQ ID NO 46 <211>LENGTH: 215 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 46 SerSer Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser 5 10 15 SerAla Gly Gly Ala Gly Gly Ser Val Ala Ser Ala Ser Gly Ala Gly 20 25 30 GluArg Arg Glu Thr Ser Leu Gly Pro Arg Ala Ala Ala Pro Arg Gly 35 40 45 ProArg Lys Cys Ala Arg Lys Thr Arg His Ala Glu Gly Gly Pro Glu 50 55 60 ProGly Ala Arg Asp Pro Ala Pro Gly Leu Thr Arg Tyr Leu Pro Ile 65 70 75 80Ala Gly Val Ser Ser Val Val Ala Leu Ala Pro Tyr Val Asn Lys Thr 85 90 95Val Thr Gly Asp Cys Leu Pro Val Leu Asp Met Glu Thr Gly His Ile 100 105110 Gly Ala Tyr Val Val Leu Val Asp Gln Thr Gly Asn Val Ala Asp Leu 115120 125 Leu Arg Ala Ala Ala Pro Ala Trp Ser Arg Arg Thr Leu Leu Pro Glu130 135 140 His Ala Arg Asn Cys Val Arg Pro Pro Asp Tyr Pro Thr Pro ProAla 145 150 155 160 Ser Glu Trp Asn Ser Leu Trp Met Thr Pro Val Gly AsnMet Leu Phe 165 170 175 Asp Gln Gly Thr Leu Val Gly Ala Leu Asp Phe HisGly Leu Arg Ser 180 185 190 Arg His Pro Trp Ser Arg Glu Gln Gly Ala ProAla Pro Ala Gly Asp 195 200 205 Ala Pro Ala Gly His Gly Glu 210 215<210> SEQ ID NO 47 <211> LENGTH: 826 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 47 Met Glu Pro Arg Pro Gly Thr Ser Ser Arg Ala AspPro Gly Pro Glu 5 10 15 Arg Pro Pro Arg Gln Thr Pro Gly Thr Gln Pro AlaAla Pro His Ala 20 25 30 Trp Gly Met Leu Asn Asp Met Gln Trp Leu Ala SerSer Asp Ser Glu 35 40 45 Glu Glu Thr Glu Val Gly Ile Ser Asp Asp Asp LeuHis Arg Asp Ser 50 55 60 Thr Ser Glu Ala Gly Ser Thr Asp Thr Glu Met PheGlu Ala Gly Leu 65 70 75 80 Met Asp Ala Ala Thr Pro Pro Ala Arg Pro ProAla Glu Arg Gln Gly 85 90 95 Ser Pro Thr Pro Ala Asp Ala Gln Gly Ser CysGly Gly Gly Pro Val 100 105 110 Gly Glu Glu Glu Ala Glu Ala Gly Gly GlyGly Asp Val Cys Ala Val 115 120 125 Cys Thr Asp Glu Ile Ala Pro Pro LeuArg Cys Gln Ser Phe Pro Cys 130 135 140 Leu His Pro Phe Cys Ile Pro CysMet Lys Thr Trp Ile Pro Leu Arg 145 150 155 160 Asn Thr Cys Pro Leu CysAsn Thr Pro Val Ala Tyr Leu Ile Val Gly 165 170 175 Val Thr Ala Ser GlySer Phe Ser Thr Ile Pro Ile Val Asn Asp Pro 180 185 190 Arg Thr Arg ValGlu Ala Glu Ala Ala Val Arg Ala Gly Thr Ala Val 195 200 205 Asp Phe IleTrp Thr Gly Asn Pro Arg Thr Ala Pro Arg Ser Leu Ser 210 215 220 Leu GlyGly His Thr Val Arg Ala Leu Ser Pro Thr Pro Pro Trp Pro 225 230 235 240Gly Thr Asp Asp Glu Asp Asp Asp Leu Ala Asp Gly Val Asp Tyr Val 245 250255 Pro Pro Ala Pro Arg Arg Ala Pro Arg Arg Gly Gly Gly Gly Ala Gly 260265 270 Ala Thr Arg Gly Thr Ser Gln Pro Ala Ala Thr Arg Pro Ala Pro Pro275 280 285 Gly Ala Pro Arg Ser Ser Ser Ser Gly Gly Ala Pro Leu Arg AlaGly 290 295 300 Val Gly Ser Gly Ser Gly Gly Gly Pro Ala Val Ala Ala ValVal Pro 305 310 315 320 Arg Val Ala Ser Leu Pro Pro Ala Ala Gly Gly GlyArg Ala Gln Ala 325 330 335 Arg Arg Val Gly Glu Asp Ala Ala Ala Ala GluGly Arg Thr Pro Pro 340 345 350 Ala Arg Gln Pro Arg Ala Ala Gln Glu ProPro Ile Val Ile Ser Asp 355 360 365 Ser Pro Pro Pro Ser Pro Arg Arg ProAla Gly Pro Gly Pro Leu Ser 370 375 380 Phe Val Ser Ser Ser Ser Ala GlnVal Ser Ser Gly Pro Gly Gly Gly 385 390 395 400 Gly Leu Pro Gln Ser SerGly Arg Ala Ala Arg Pro Arg Ala Ala Val 405 410 415 Ala Pro Arg Val ArgSer Pro Pro Arg Ala Ala Ala Ala Pro Val Val 420 425 430 Ser Ala Ser AlaAsp Ala Ala Gly Pro Ala Pro Pro Ala Val Pro Val 435 440 445 Asp Ala HisArg Ala Pro Arg Ser Arg Met Thr Gln Ala Gln Thr Asp 450 455 460 Thr GlnAla Gln Ser Leu Gly Arg Ala Gly Ala Thr Asp Ala Arg Gly 465 470 475 480Ser Gly Gly Pro Gly Ala Glu Gly Gly Pro Gly Val Pro Arg Gly Thr 485 490495 Asn Thr Pro Gly Ala Ala Pro His Ala Ala Glu Gly Ala Ala Ala Arg 500505 510 Pro Arg Lys Arg Arg Gly Ser Asp Ser Gly Pro Ala Ala Ser Ser Ser515 520 525 Ala Ser Ser Ser Ala Ala Pro Arg Ser Pro Leu Ala Pro Gln GlyVal 530 535 540 Gly Ala Lys Arg Ala Ala Pro Arg Arg Ala Pro Asp Ser AspSer Gly 545 550 555 560 Asp Arg Gly His Gly Pro Leu Ala Pro Ala Ser AlaGly Ala Ala Pro 565 570 575 Pro Ser Ala Ser Pro Ser Ser Gln Ala Ala ValAla Ala Ala Ser Ser 580 585 590 Ser Ser Ala Ser Ser Ser Ser Ala Ser SerSer Ser Ala Ser Ser Ser 595 600 605 Ser Ala Ser Ser Ser Ser Ala Ser SerSer Ser Ala Ser Ser Ser Ser 610 615 620 Ala Ser Ser Ser Ala Gly Gly AlaGly Gly Ser Val Ala Ser Ala Ser 625 630 635 640 Gly Ala Gly Glu Arg ArgGlu Thr Ser Leu Gly Pro Arg Ala Ala Ala 645 650 655 Pro Arg Gly Pro ArgLys Cys Ala Arg Lys Thr Arg His Ala Glu Gly 660 665 670 Gly Pro Glu ProGly Ala Arg Asp Pro Ala Pro Gly Leu Thr Arg Tyr 675 680 685 Leu Pro IleAla Gly Val Ser Ser Val Val Ala Leu Ala Pro Tyr Val 690 695 700 Asn LysThr Val Thr Gly Asp Cys Leu Pro Val Leu Asp Met Glu Thr 705 710 715 720Gly His Ile Gly Ala Tyr Val Val Leu Val Asp Gln Thr Gly Asn Val 725 730735 Ala Asp Leu Leu Arg Ala Ala Ala Pro Ala Trp Ser Arg Arg Thr Leu 740745 750 Leu Pro Glu His Ala Arg Asn Cys Val Arg Pro Pro Asp Tyr Pro Thr755 760 765 Pro Pro Ala Ser Glu Trp Asn Ser Leu Trp Met Thr Pro Val GlyAsn 770 775 780 Met Leu Phe Asp Gln Gly Thr Leu Val Gly Ala Leu Asp PheHis Gly 785 790 795 800 Leu Arg Ser Arg His Pro Trp Ser Arg Glu Gln GlyAla Pro Ala Pro 805 810 815 Ala Gly Asp Ala Pro Ala Gly His Gly Glu 820825 <210> SEQ ID NO 48 <211> LENGTH: 3350 <212> TYPE: DNA <213>ORGANISM: HSV-2 <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: 1027, 1034, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061,1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073,1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085,1086, 1087, 1088, 1089, 1090 <223> OTHER INFORMATION: n = A,T,C or G<221> NAME/KEY: misc_feature <222> LOCATION: 1091, 1092, 1093, 1094,1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106,1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118,1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129 <223>OTHER INFORMATION: n = A,T,C or G <221> NAME/KEY: misc_feature <222>LOCATION: 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139,1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151,1152, 1327, 1364, 1390, 1392 <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 48 ccgtcggtga cctgcaggag ctcgtttatt aatagccagtccatgctcag cgtagcggcc 60 agcccctggg gagacaggtc cacggagtcc ggaaccaccgtcggctgacc caggggcccc 120 aggctgtagt ccccccaggc ccccaggtca tgacggttcgtgagcacgac gaggtctgcg 180 gccgggctgg ggggcgcgtc ctcggtcgcg tgggccatcacctcctgaat ggctgcggtg 240 cgctgatcgg ccgagctggc gaagggcgcc acgaccagcgcgcgctccgt ctgcaggccc 300 ttccacgtgt cgtggagttc ctgaacgaac tcggccacccgctcggggcc cgtggccgcg 360 cgcgcggcct gatagccggc cgagaggcgc cgccagcgcgccaggaactg actcatgtaa 420 cagaacccgg ggacctggtc ccccgacatc aactttgacgccctggcgtg gatgcccgac 480 acgatggcca ggaacccgtg gatttcccgc cgcacgacggccagcacgtt accctcgtgc 540 gagacctggg ccgccagctc gtcgcatacc ccgaggtgcgccgtcgtctc ggtgacgacg 600 gaccgcagcc ccgcgaggga cgcgaccagc gcgcgcttggcgtcgtgata catgccgcag 660 tactggctca ccgcgtcgcc catggcctcg gggcgccagggccccaggcg ctcgtgggcg 720 tctgcgacca cggcgtacag gcggtgcccg tcgctctcgaaccggcactc aaagaaggcg 780 gcgagcgtgc gcatgtgaag ccgcagcagc acgatcgcgtcctccagctg gcggaccagg 840 gggtcggcgc gctcggcgag ctcctgcagc accccccgggccgccagggc gtacatgctg 900 atcagcagca ggctgctgcc cacctcggga ggctgggggggaggcagctg gaccgcgggc 960 cgcagctgct cgacggcccc cctggcgatc acgtacagctcgcgcagcag ctgctcgatg 1020 ttgtcgngcc atcntgcatc gtgggcccga cgcnnnnnnnnnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 1140 nnnnnnnnnn nnaggccagc acccgcaggg caaactcgatggggcggggc aggtaggcag 1200 cgttgcacgt ggccctcagc gcgtccccga ccaccagggccagcacgtaa gggacgaacc 1260 ccgggtcggc gaggacgttg gggtggatgc cctccagggccgggaagcgg atcttggtgg 1320 ccgcggncag gtgaaccgag ggggcgtggc taggcggcccgacngggagc atcgcggaca 1380 gcggcgtggn cngggtggtg ggggtcaggt cccagtgggtctggccgtac acgtcgagcc 1440 agatgagcgc cgtctcgcgc aggaggctgg gctggccggcgctgaagcgg cgctcggccg 1500 tctcaaactc ccccacgagc gtgcgccgca ggctcgccaggtgttccgtc ggcacggccg 1560 ggcccatgat gcgcgccagc gtctggctga ggacgccgcccgacaggccg accgcctcac 1620 agagccgccc gtgcgtgtgc tcgctggcgc cctggatccgccggaacgtt ttcacgtagc 1680 cggcgtagtg cccgtactcc cgcgcgagcc cgaacacgttcgcccccgca agggcaatgc 1740 acccaaagag ctgctggatc tcgctgagcc cgtggccggggggcgtccgc gcgggcaccc 1800 ccgccaccaa aaacccctcc agggccgata tgtactgggtgcagtgcgcg ggcgtgaacc 1860 ccgcgtcggt aagcgtgttg atcaccacgg agggcgagttgctgttctgg accaaagccc 1920 acgtctgctg cagcagcgcg aggagccgtt gctgggccccggcggagggc ggctccccta 1980 gctgcagcag gccggtgacg gccggacgga agatggccagcgccgacgca ctcagaaacg 2040 gcacgtcggg gtcgaagacg gccgcgtccg tccgcacgcgcgccatcagc gtccccgggg 2100 gcgcgcacgc cgaccgcggg ctgacgcggc ttagggcggtcgacacgcgc acctcctcgc 2160 gactgcgaac cattttggtg gcctcgaggg gcgggatcatgatagccggg tcgatctccc 2220 gcaccgtgtg ctgaaactgg gccagcagcg gcggcgggaccaccgcgccc cgatcggggg 2280 tcgtcaggta ctcgtccacc agcgccagcg taaacagggcccgcgtgagg ggggtcaggg 2340 cggcgtcgtc gatgcgctgt aggtgcgccg agaacagcgtcacccaattg ctgaccaggg 2400 ccaagaaccg gagaccctct tgcacgatcg gggacgggaagagcaggctg tacgccgggg 2460 tggtcaggtt ggcgccgggt tgccccaggg gaaccggggacatcttaagc gacatctccc 2520 cgagggcctc cagggaggtc cgcgggttca tggccaggcagctctgggtg acggtccgcc 2580 agcggtcgat ccactccacg gcacactggc ggacgcgcaccggccccagg gccgccgtgg 2640 tgcgcagccc ggcggcctcc agcgcgtggg tcgtgtcggagccggtgatc gccaggaccg 2700 tgtccttgat gacgtccatc tcccggaagg ccgcctcgggggtctcgggg agcgccaccg 2760 ccatgcggtg caccagcagc ccggggaggt tctcggccaagagcgccgtc tccggaagcc 2820 cgtgggcccg gtgcaaggcg cacagttgct ccaggagcgggtgccagcac gcccgcgcct 2880 ccgccgggcc gaccgccgcg cccgacaaca gaaacgccgccgtggcggcg cgcagtttgg 2940 ccgcggacag aaacgccggc tcgtccgcgc tgcccgccggctcgctcgag ggggagggcg 3000 gccggcggag gttggtcagg ctccccaaca ggacctgcaacggtccgttt gggggtggag 3060 cggacggggg ggtcatgccg gcgggcgccg ggacctggagcgcgctgtcc gacatggcga 3120 ccggcgtgcg cgctcggcga cgcggcgcgg agaccgcgggcccaaacggg aatgactgcc 3180 gccgccctat acggaggggc taagtatcgc ccggggacccttcgaaaccc cgggcgtgtc 3240 gcaagtacgc cgcgaaggcg cggcgtgtta tacggcgcgttatgtcccgg cattccgttc 3300 gtgggttcgg gcccgggtgc tgtcgggtgg gagtgtgtgtgtgtgggggg 3350 <210> SEQ ID NO 49 <211> LENGTH: 3345 <212> TYPE: DNA<213> ORGANISM: HSV-2 <400> SEQUENCE: 49 atgtcggaca gcgcgctccaggtcccggcg cccgccggca tgaccccccc gtccgctcca 60 cccccaaacg gaccgttgcaggtcctgttg gggagcctga ccaacctccg ccggccgccc 120 tccccctcga gcgagccggcgggcagcgcg gacgagccgg cgtttctgtc cgcggccaaa 180 ctgcacgccg ccacggcggcgtttctgttg tcgggcgcgg cggtcggccc ggcggaggcg 240 cgggcgtgct ggcacccgctcctggagcaa ctgtgcgcct tgcaccgggc ccacgggctt 300 ccggagacgg cgctcttggccgagaacctc cccgggctgc tggtgcaccg catggcggtg 360 gcgctccccg agacccccgaggcggccttc cgggagatgg acgtcatcaa ggacacggtc 420 ctggcgatca ccggctccgacacgacccac gcgctggagg ccgccgggct gcgcaccacg 480 gcggccctgg ggccggtgcgcgtccgccag tgtgccgtgg agtggatcga ccgctggcgg 540 accgtcaccc agagctgcctggccatgaac ccgcggacct ccctggaggc cctcggggag 600 atgtcgctta agatgtccccggttcccctg gggcaacccg gcgccaacct gaccaccccg 660 gcgtacagcc tgctcttcccgtccccgatc gtgcaagagg gtctccggtt cttggccctg 720 gtcagcaatt gggtgacgctgttctcggcg cacctacagc gcatcgacga cgccgccctg 780 acccccctca cgcgggccctgtttacgctg gcgctggtgg acgactacct gacgaccccc 840 gatcggggcg cggtggtcccgccgccgctg ctggcccagt ttcagcacac ggtgcgggag 900 atcgacccgg ctatcatgatcccgcccctc gaggccacca aaatggttcg cagtcgcgag 960 gaggtgcgcg tgtcgaccgccctaagccgc gtcagcccgc ggtcggcgtg cgcgcccccg 1020 gggacgctga tggcgcgcgtgcggacggac gcggccgtct tcgaccccga cgtgccgttt 1080 ctgagtgcgt cggcgctggccatcttccgt ccggccgtca ccggcctgct gcagctaggg 1140 gagccgccct ccgccggggcccagcaacgg ctcctcgcgc tgctgcagca gacgtgggct 1200 ttggtccaga acagcaactcgccctccgtg gtgatcaaca cgcttaccga cgcggggttc 1260 acgcccgcgc actgcacccagtacatatcg gccctggagg ggtttttggt ggcgggggtg 1320 cccgcgcgga cgccccccggccacgggctc agcgagatcc agcagctctt tgggtgcatt 1380 gcccttgcgg gggcgaacgtgttcgggctc gcgcgggagt acgggcacta cgccggctac 1440 gtgaaaacgt tccggcggatccagggcgcc agcgagcaca cgcacgggcg gctctgtgag 1500 gcggtcggcc tgtcgggcggcgtcctcagc cagacgctgg cgcgcatcat gggcccggcc 1560 gtgccgacgg aacacctggcgagcctgcgg cgcacgctcg tgggggagtt tgagacggcc 1620 gagcgccgct tcagcgccggccagcccagc ctcctgcgcg agacggcgct catctggctc 1680 gacgtgtacg gccagacccactgggacctg acccccacca ccccggccac gccgctgtcc 1740 gcgctgctcc ccgtcgggccgcctagccac gccccctcgg ttcacctggc cgcggccacc 1800 aagatccgct tcccggccctggagggcatc caccccaacg tcctcgccga cccggggttc 1860 gtcccttacg tgctggccctggtggtcggg gacgcgctga gggccacgtg caacgctgcc 1920 tacctgcccc gccccatcgagtttgccctg cgggtgctgg cctgggcgcg cgacttcggc 1980 ctgggctatc tccccaccgtcgaggggcac cgcacaaaat tgggcgcgct gatcaccctc 2040 ctcgaaccgg ccacccgggccggcgtcggg cccacgatgc agatggccga caacatcgag 2100 cagctgctgc gcgagctgtacgtgatcgcc aggggggccg tcgagcagct gcggcccgcg 2160 gtccagctgc ctcccccccagcctcccgag gtgggcagca gcctgctgct gatcagcatg 2220 tacgccctgg cggcccggggggtgctgcag gagctcgccg agcgcgccga ccccctggtc 2280 cgccagctgg aggacgcgatcgtgctgctg cggctgcaca tgcgcacgct cgccgccttc 2340 tttgagtgcc ggttcgagagcgacgggcac cgcctgtacg ccgtggtcgc agacgcccac 2400 gagcgcctgg ggccctggcgccccgaggcc atgggcgacg cggtgagcca gtactgcggc 2460 atgtatcacg acgccaagcgcgcgctggtc gcgtccctcg cggggctgcg gtccgtcgtc 2520 accgagacga cggcgcacctcggggtatgc gacgagctgg cggcccaggt ctcgcacgag 2580 ggtaacgtgc tggccgtcgtgcggcgggaa atccacgggt tcctggccat cgtgtcgggc 2640 atccacgcca gggcgtcaaagttgatgtcg ggggaccagg tccccgggtt ctgttacatg 2700 agtcagttcc tggcgcgctggcggcgcctc tcggccggct atcaggccgc acgcgcggcc 2760 acgggccccg agcgggtggccgagttcgtt caggaactcc acgacacgtg gaagggcctg 2820 cagacggagc gcgcgctggtcgtggcgcgc ttcgccagct cggccgatca gcgcaccgca 2880 gccattcagg aggtgatggcccacgcgacc gaggacgcgc cccccagccc ggccgcagac 2940 ctcgtcgtgc tcacgaaccgtcatgacctg ggggcctggg gggactacag cctggggccc 3000 ctgggtcagc cgacggtggttccggactcc gtggacctgt ctccccaggg gctggccgct 3060 acgctgagca tggactggctattaataaac gagctcctgc aggtcaccga cggcgtgttt 3120 cgcgcctcgg cgtttcggccttccgccggc ccgggggccc ccggggacct ggaggcccaa 3180 gatgccggcg gtagcacccccgaacccacg acacccggcc cacaggacac gcaggcccgc 3240 gcgccgtcga cgcgcccggcgggccgcgag acggtccctt ggcccaacac ccccgtggag 3300 gacgacgaga tgacgccgcaggagacacca ccggtacacc cgtag 3345 <210> SEQ ID NO 50 <211> LENGTH: 993<212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 50 Glu Pro Ala GlySer Ala Asp Glu Pro Ala Phe Leu Ser Ala Ala Lys 5 10 15 Leu His Ala AlaThr Ala Ala Phe Leu Leu Ser Gly Ala Ala Val Gly 20 25 30 Pro Ala Glu AlaArg Ala Cys Trp His Pro Leu Leu Glu Gln Leu Cys 35 40 45 Ala Leu His ArgAla His Gly Leu Pro Glu Thr Ala Leu Leu Ala Glu 50 55 60 Asn Leu Pro GlyLeu Leu Val His Arg Met Ala Val Ala Leu Pro Glu 65 70 75 80 Thr Pro GluAla Ala Phe Arg Glu Met Asp Val Ile Lys Asp Thr Val 85 90 95 Leu Ala IleThr Gly Ser Asp Thr Thr His Ala Leu Glu Ala Ala Gly 100 105 110 Leu ArgThr Thr Ala Ala Leu Gly Pro Val Arg Val Arg Gln Cys Ala 115 120 125 ValGlu Trp Ile Asp Arg Trp Arg Thr Val Thr Gln Ser Cys Leu Ala 130 135 140Met Asn Pro Arg Thr Ser Leu Glu Ala Leu Gly Glu Met Ser Leu Lys 145 150155 160 Met Ser Pro Val Pro Leu Gly Gln Pro Gly Ala Asn Leu Thr Thr Pro165 170 175 Ala Tyr Ser Leu Leu Phe Pro Ser Pro Ile Val Gln Glu Gly LeuArg 180 185 190 Phe Leu Ala Leu Val Ser Asn Trp Val Thr Leu Phe Ser AlaHis Leu 195 200 205 Gln Arg Ile Asp Asp Ala Ala Leu Thr Pro Leu Thr ArgAla Leu Phe 210 215 220 Thr Leu Ala Leu Val Asp Asp Tyr Leu Thr Thr ProAsp Arg Gly Ala 225 230 235 240 Val Val Pro Pro Pro Leu Leu Ala Gln PheGln His Thr Val Arg Glu 245 250 255 Ile Asp Pro Ala Ile Met Ile Pro ProLeu Glu Ala Thr Lys Met Val 260 265 270 Arg Ser Arg Glu Glu Val Arg ValSer Thr Ala Leu Ser Arg Val Ser 275 280 285 Pro Arg Ser Ala Cys Ala ProPro Gly Thr Leu Met Ala Arg Val Arg 290 295 300 Thr Asp Ala Ala Val PheAsp Pro Asp Val Pro Phe Leu Ser Ala Ser 305 310 315 320 Ala Leu Ala IlePhe Arg Pro Ala Val Thr Gly Leu Leu Gln Leu Gly 325 330 335 Glu Pro ProSer Ala Gly Ala Gln Gln Arg Leu Leu Ala Leu Leu Gln 340 345 350 Gln ThrTrp Ala Leu Val Gln Asn Ser Asn Ser Pro Ser Val Val Ile 355 360 365 AsnThr Leu Thr Asp Ala Gly Phe Thr Pro Ala His Cys Thr Gln Tyr 370 375 380Ile Ser Ala Leu Glu Gly Phe Leu Val Ala Gly Val Pro Ala Arg Thr 385 390395 400 Pro Pro Gly His Gly Leu Ser Glu Ile Gln Gln Leu Phe Gly Cys Ile405 410 415 Ala Leu Ala Gly Ala Asn Val Phe Gly Leu Ala Arg Glu Tyr GlyHis 420 425 430 Tyr Ala Gly Tyr Val Lys Thr Phe Arg Arg Ile Gln Gly AlaSer Glu 435 440 445 His Thr His Gly Arg Leu Cys Glu Ala Val Gly Leu SerGly Gly Val 450 455 460 Leu Ser Gln Thr Leu Ala Arg Ile Met Gly Pro AlaVal Pro Thr Glu 465 470 475 480 His Leu Ala Ser Leu Arg Arg Thr Leu ValGly Glu Phe Glu Thr Ala 485 490 495 Glu Arg Arg Phe Ser Ala Gly Gln ProSer Leu Leu Arg Glu Thr Ala 500 505 510 Leu Ile Trp Leu Asp Val Tyr GlyGln Thr His Trp Asp Leu Thr Pro 515 520 525 Thr Thr Pro Ala Thr Pro LeuSer Ala Leu Leu Pro Val Gly Pro Pro 530 535 540 Ser His Ala Pro Ser ValHis Leu Ala Ala Ala Thr Lys Ile Arg Phe 545 550 555 560 Pro Ala Leu GluGly Ile His Pro Asn Val Leu Ala Asp Pro Gly Phe 565 570 575 Val Pro TyrVal Leu Ala Leu Val Val Gly Asp Ala Leu Arg Ala Thr 580 585 590 Cys AsnAla Ala Tyr Leu Pro Arg Pro Ile Glu Phe Ala Leu Arg Val 595 600 605 LeuAla Trp Ala Arg Asp Phe Gly Leu Gly Tyr Leu Pro Thr Val Glu 610 615 620Gly His Arg Thr Lys Leu Gly Ala Leu Ile Thr Leu Leu Glu Pro Ala 625 630635 640 Thr Arg Ala Gly Val Gly Pro Thr Met Gln Met Ala Asp Asn Ile Glu645 650 655 Gln Leu Leu Arg Glu Leu Tyr Val Ile Ala Arg Gly Ala Val GluGln 660 665 670 Leu Arg Pro Ala Val Gln Leu Pro Pro Pro Gln Pro Pro GluVal Gly 675 680 685 Ser Ser Leu Leu Leu Ile Ser Met Tyr Ala Leu Ala AlaArg Gly Val 690 695 700 Leu Gln Glu Leu Ala Glu Arg Ala Asp Pro Leu ValArg Gln Leu Glu 705 710 715 720 Asp Ala Ile Val Leu Leu Arg Leu His MetArg Thr Leu Ala Ala Phe 725 730 735 Phe Glu Cys Arg Phe Glu Ser Asp GlyHis Arg Leu Tyr Ala Val Val 740 745 750 Ala Asp Ala His Glu Arg Leu GlyPro Trp Arg Pro Glu Ala Met Gly 755 760 765 Asp Ala Val Ser Gln Tyr CysGly Met Tyr His Asp Ala Lys Arg Ala 770 775 780 Leu Val Ala Ser Leu AlaGly Leu Arg Ser Val Val Thr Glu Thr Thr 785 790 795 800 Ala His Leu GlyVal Cys Asp Glu Leu Ala Ala Gln Val Ser His Glu 805 810 815 Gly Asn ValLeu Ala Val Val Arg Arg Glu Ile His Gly Phe Leu Ala 820 825 830 Ile ValSer Gly Ile His Ala Arg Ala Ser Lys Leu Met Ser Gly Asp 835 840 845 GlnVal Pro Gly Phe Cys Tyr Met Ser Gln Phe Leu Ala Arg Trp Arg 850 855 860Arg Leu Ser Ala Gly Tyr Gln Ala Ala Arg Ala Ala Thr Gly Pro Glu 865 870875 880 Arg Val Ala Glu Phe Val Gln Glu Leu His Asp Thr Trp Lys Gly Leu885 890 895 Gln Thr Glu Arg Ala Leu Val Val Ala Arg Phe Ala Ser Ser AlaAsp 900 905 910 Gln Arg Thr Ala Ala Ile Gln Glu Val Met Ala His Ala ThrGlu Asp 915 920 925 Ala Pro Pro Ser Pro Ala Ala Asp Leu Val Val Leu ThrAsn Arg His 930 935 940 Asp Leu Gly Ala Trp Gly Asp Tyr Ser Leu Gly ProLeu Gly Gln Pro 945 950 955 960 Thr Val Val Pro Asp Ser Val Asp Leu SerPro Gln Gly Leu Ala Ala 965 970 975 Thr Leu Ser Met Asp Trp Leu Leu IleAsn Glu Leu Leu Gln Val Thr 980 985 990 Asp <210> SEQ ID NO 51 <211>LENGTH: 1113 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 51Met Ser Asp Ser Ala Leu Gln Val Pro Ala Pro Ala Gly Met Thr Pro 5 10 15Pro Ser Ala Pro Pro Pro Asn Gly Pro Leu Gln Val Leu Leu Gly Ser 20 25 30Leu Thr Asn Leu Arg Arg Pro Pro Ser Pro Ser Ser Glu Pro Ala Gly 35 40 45Ser Ala Asp Glu Pro Ala Phe Leu Ser Ala Ala Lys Leu His Ala Ala 50 55 60Thr Ala Ala Phe Leu Leu Ser Gly Ala Ala Val Gly Pro Ala Glu Ala 65 70 7580 Arg Ala Cys Trp His Pro Leu Leu Glu Gln Leu Cys Ala Leu His Arg 85 9095 Ala His Gly Leu Pro Glu Thr Ala Leu Leu Ala Glu Asn Leu Pro Gly 100105 110 Leu Leu Val His Arg Met Ala Val Ala Leu Pro Glu Thr Pro Glu Ala115 120 125 Ala Phe Arg Glu Met Asp Val Ile Lys Asp Thr Val Leu Ala IleThr 130 135 140 Gly Ser Asp Thr Thr His Ala Leu Glu Ala Ala Gly Leu ArgThr Thr 145 150 155 160 Ala Ala Leu Gly Pro Val Arg Val Arg Gln Cys AlaVal Glu Trp Ile 165 170 175 Asp Arg Trp Arg Thr Val Thr Gln Ser Cys LeuAla Met Asn Pro Arg 180 185 190 Thr Ser Leu Glu Ala Leu Gly Glu Met SerLeu Lys Met Ser Pro Val 195 200 205 Pro Leu Gly Gln Pro Gly Ala Asn LeuThr Thr Pro Ala Tyr Ser Leu 210 215 220 Leu Phe Pro Ser Pro Ile Val GlnGlu Gly Leu Arg Phe Leu Ala Leu 225 230 235 240 Val Ser Asn Trp Val ThrLeu Phe Ser Ala His Leu Gln Arg Ile Asp 245 250 255 Asp Ala Ala Leu ThrPro Leu Thr Arg Ala Leu Phe Thr Leu Ala Leu 260 265 270 Val Asp Asp TyrLeu Thr Thr Pro Asp Arg Gly Ala Val Val Pro Pro 275 280 285 Pro Leu LeuAla Gln Phe Gln His Thr Val Arg Glu Ile Asp Pro Ala 290 295 300 Ile MetIle Pro Pro Leu Glu Ala Thr Lys Met Val Arg Ser Arg Glu 305 310 315 320Glu Val Arg Val Ser Thr Ala Leu Ser Arg Val Ser Pro Arg Ser Ala 325 330335 Cys Ala Pro Pro Gly Thr Leu Met Ala Arg Val Arg Thr Asp Ala Ala 340345 350 Val Phe Asp Pro Asp Val Pro Phe Leu Ser Ala Ser Ala Leu Ala Ile355 360 365 Phe Arg Pro Ala Val Thr Gly Leu Leu Gln Leu Gly Glu Pro ProSer 370 375 380 Ala Gly Ala Gln Gln Arg Leu Leu Ala Leu Leu Gln Gln ThrTrp Ala 385 390 395 400 Leu Val Gln Asn Ser Asn Ser Pro Ser Val Val IleAsn Thr Leu Thr 405 410 415 Asp Ala Gly Phe Thr Pro Ala His Cys Thr GlnTyr Ile Ser Ala Leu 420 425 430 Glu Gly Phe Leu Val Ala Gly Val Pro AlaArg Thr Pro Pro Gly His 435 440 445 Gly Leu Ser Glu Ile Gln Gln Leu PheGly Cys Ile Ala Leu Ala Gly 450 455 460 Ala Asn Val Phe Gly Leu Ala ArgGlu Tyr Gly His Tyr Ala Gly Tyr 465 470 475 480 Val Lys Thr Phe Arg ArgIle Gln Gly Ala Ser Glu His Thr His Gly 485 490 495 Arg Leu Cys Glu AlaVal Gly Leu Ser Gly Gly Val Leu Ser Gln Thr 500 505 510 Leu Ala Arg IleMet Gly Pro Ala Val Pro Thr Glu His Leu Ala Ser 515 520 525 Leu Arg ArgThr Leu Val Gly Glu Phe Glu Thr Ala Glu Arg Arg Phe 530 535 540 Ser AlaGly Gln Pro Ser Leu Leu Arg Glu Thr Ala Leu Ile Trp Leu 545 550 555 560Asp Val Tyr Gly Gln Thr His Trp Asp Leu Thr Pro Thr Thr Pro Ala 565 570575 Thr Pro Leu Ser Ala Leu Leu Pro Val Gly Pro Pro Ser His Ala Pro 580585 590 Ser Val His Leu Ala Ala Ala Thr Lys Ile Arg Phe Pro Ala Leu Glu595 600 605 Gly Ile His Pro Asn Val Leu Ala Asp Pro Gly Phe Val Pro TyrVal 610 615 620 Leu Ala Leu Val Val Gly Asp Ala Leu Arg Ala Thr Cys AsnAla Ala 625 630 635 640 Tyr Leu Pro Arg Pro Ile Glu Phe Ala Leu Arg ValLeu Ala Trp Ala 645 650 655 Arg Asp Phe Gly Leu Gly Tyr Leu Pro Thr ValGlu Gly His Arg Thr 660 665 670 Lys Leu Gly Ala Leu Ile Thr Leu Leu GluPro Ala Thr Arg Ala Gly 675 680 685 Val Gly Pro Thr Met Gln Met Ala AspAsn Ile Glu Gln Leu Leu Arg 690 695 700 Glu Leu Tyr Val Ile Ala Arg GlyAla Val Glu Gln Leu Arg Pro Ala 705 710 715 720 Val Gln Leu Pro Pro ProGln Pro Pro Glu Val Gly Ser Ser Leu Leu 725 730 735 Leu Ile Ser Met TyrAla Leu Ala Ala Arg Gly Val Leu Gln Glu Leu 740 745 750 Ala Glu Arg AlaAsp Pro Leu Val Arg Gln Leu Glu Asp Ala Ile Val 755 760 765 Leu Leu ArgLeu His Met Arg Thr Leu Ala Ala Phe Phe Glu Cys Arg 770 775 780 Phe GluSer Asp Gly His Arg Leu Tyr Ala Val Val Ala Asp Ala His 785 790 795 800Glu Arg Leu Gly Pro Trp Arg Pro Glu Ala Met Gly Asp Ala Val Ser 805 810815 Gln Tyr Cys Gly Met Tyr His Asp Ala Lys Arg Ala Leu Val Ala Ser 820825 830 Leu Ala Gly Leu Arg Ser Val Val Thr Glu Thr Thr Ala His Leu Gly835 840 845 Val Cys Asp Glu Leu Ala Ala Gln Val Ser His Glu Gly Asn ValLeu 850 855 860 Ala Val Val Arg Arg Glu Ile His Gly Phe Leu Ala Ile ValSer Gly 865 870 875 880 Ile His Ala Arg Ala Ser Lys Leu Met Ser Gly AspGln Val Pro Gly 885 890 895 Phe Cys Tyr Met Ser Gln Phe Leu Ala Arg TrpArg Arg Leu Ser Ala 900 905 910 Gly Tyr Gln Ala Ala Arg Ala Ala Thr GlyPro Glu Arg Val Ala Glu 915 920 925 Phe Val Gln Glu Leu His Asp Thr TrpLys Gly Leu Gln Thr Glu Arg 930 935 940 Ala Leu Val Val Ala Arg Phe AlaSer Ser Ala Asp Gln Arg Thr Ala 945 950 955 960 Ala Ile Gln Glu Val MetAla His Ala Thr Glu Asp Ala Pro Pro Ser 965 970 975 Pro Ala Ala Asp LeuVal Val Leu Thr Asn Arg His Asp Leu Gly Ala 980 985 990 Trp Gly Asp TyrSer Leu Gly Pro Leu Gly Gln Pro Thr Val Val Pro 995 1000 1005 Asp SerVal Asp Leu Ser Pro Gln Gly Leu Ala Ala Thr Leu Ser Met 1010 1015 1020Asp Trp Leu Leu Ile Asn Glu Leu Leu Gln Val Thr Asp Gly Val Phe 10251030 1035 1040 Arg Ala Ser Ala Phe Arg Pro Ser Ala Gly Pro Gly Ala ProGly Asp 1045 1050 1055 Leu Glu Ala Gln Asp Ala Gly Gly Ser Thr Pro GluPro Thr Thr Pro 1060 1065 1070 Gly Pro Gln Asp Thr Gln Ala Arg Ala ProSer Thr Pro Ala Gly Arg 1075 1080 1085 Glu Thr Val Pro Trp Pro Asn ThrPro Val Glu Asp Asp Glu Met Thr 1090 1095 1100 Pro Gln Glu Thr Pro ProVal His Pro 1105 1110 <210> SEQ ID NO 52 <211> LENGTH: 3113 <212> TYPE:DNA <213> ORGANISM: HSV-2 <400> SEQUENCE: 52 atgtcggaca gcgcgctccaggtcccggcg cccgccggca tgaccccccc gtccgctcca 60 cccccaaacg gaccgttgcaggtcctgttg gggagcctga ccaacctccg ccggccgccc 120 tccccctcga gcgagccggcgggcagcgcg gacgagccgg cgtttctgtc cgcggccaaa 180 ctgcgcgccg ccacggcggcgtttctgttg tcgggcgcgg cggtcggccc ggcggaggcg 240 cgggcgtgct ggcacccgctcctggagcaa ctgtgcgcct tgcaccgggc ccacgggctt 300 ccggagacgg cgctcttggccgagaacctc cccgggctgc tggtgcaccg catggcggtg 360 gcgctccccg agacccccgaggcggccttc cgggagatgg acgtcatcaa ggacacggtc 420 ctggcgatca ccggctccgacacgacccac gcgctggagg ccgccgggct gcgcaccacg 480 gcggccctgg ggccggtgcgcgtccgccag tgtgccgtgg agtggatcga ccgctggcgg 540 accgtcaccc agagctgcctggccatgaac ccgcggacct ccctggaggc cctcggggag 600 atgtcgctta agatgtccccggttcccctg gggcaacccg gcgccaacct gaccaccccg 660 gcgtacagcc tgctcttcccgtccccgatc gtgcaagagg gtctccggtt cttggccctg 720 gtcagcaatt gggtgacgctgttctcggcg cacctacagc gcatcgacga cgccgccctg 780 acccccctca cgcgggccctgtttacgctg gcgctggtgg acgagtacct gacgaccccc 840 gatcggggcg cggtggtcccgccgccgctg ctggcccagt ttcagcacac ggtgcgggag 900 atcgacccgg ctatcatgatcccgcccctc gaggccacca aaatggttcg cagtcgcgag 960 gaggtgcgcg tgtcgaccgccctaagccgc gtcagcccgc ggtcggcgtg cgcgcccccg 1020 gggacgctga tggcgcgcgtgcggacggac gcggccgtct tcgaccccga cgtgccgttt 1080 ctgagtgcgt cggcgctggccatcttccgt ccggccgtca ccggcctgct gcagctaggg 1140 gagccgccct ccgccggggcccagcaacgg ctcctcgcgc tgctgcagca gacgtgggct 1200 ttggtccaga acagcaactcgccctccgtg gtgatcaaca cgcttaccga cgcggggttc 1260 acgcccgcgc actgcacccagtacatatcg gccctggagg ggtttttggt ggcgggggtg 1320 cccgcgcgga cgccccccggccacgggctc agcgagatcc agcagctctt tgggtgcatt 1380 gcccttgcgg gggcgaacgtgttcgggctc gcgcgggagt acgggcacta cgccggctac 1440 gtgaaaacgt tccggcggatccagggcgcc agcgagcaca cgcacgggcg gctctgtgag 1500 gcggtcggcc tgtcgggcggcgtcctcagc cagacgctgg cgcgcatcat gggcccggcc 1560 gtgccgacgg aacacctggcgagcctgcgg cgcacgctcg tgggggagtt tgagacggcc 1620 gagcgccgct tcagcgccggccagcccagc ctcctgcgcg agacggcgct catctggctc 1680 gacgtgtacg gccagacccactgggacctg acccccacca ccccggccac gccgctgtcc 1740 gcgctgctcc ccgtcgggccgcctagccac gccccctcgg ttcacctggc cgcggccacc 1800 aagatccgct tcccggccctggagggcatc caccccaacg tcctcgccga cccggggttc 1860 gtcccttacg tgctggccctggtggtcggg gacgcgctga gggccacgtg caacgctgcc 1920 tacctgcccc gccccatcgagtttgccctg cgggtgctgg cctgggcgcg cgacttcggc 1980 ctgggctatc tccccaccgtcgaggggcac cgcacaaaat tgggcgcgct gatcaccctc 2040 ctcgaaccgg ccacccgggccggcgtcggg cccacgatgc agatggccga caacatcgag 2100 cagctgctgc gcgagctgtacgtgatcgcc aggggggccg tcgagcagct gcggcccgcg 2160 gtccagctgc ctcccccccagcctcccgag gtgggcagca gcctgctgct gatcagcatg 2220 tacgccctgg cggcccggggggtgctgcag gagctcgccg agcgcgccga ccccctggtc 2280 cgccagctgg aggacgcgatcgtgctgctg cggcttcaca tgcgcacgct cgccgccttc 2340 tttgagtgcc ggttcgagagcgacgggcac cgcctgtacg ccgtggtcgc agacgcccac 2400 gagcgcctgg ggccctggcgccccgaggcc atgggcgacg cggtgagcca gtactgcggc 2460 atgtatcacg acgccaagcgcgcgctggtc gcgtccctcg cggggctgcg gtccgtcgtc 2520 accgagacga cggcgcacctcggggtatgc gacgagctgg cggcccaggt ctcgcacgag 2580 ggtaacgtgc tggccgtcgtgcggcgggaa atccacgggt tcctggccat cgtgtcgggc 2640 atccacgcca gggcgtcaaagttgatgtcg ggggaccagg tccccgggtt ctgttacatg 2700 agtcagttcc tggcgcgctggcggcgcctc tcggccggct atcaggccgc gcgcgcggcc 2760 acgggccccg agcgggtggccgagttcgtt caggaactcc acgacacgtg gaagggcctg 2820 cagacggagc gcgcgctggtcgtggcgccc ttcgccagct cggccgatca gcgcaccgca 2880 gccattcagg aggtgatggcccacgcgacc gaggacgcgc cccccagccc ggccgcagac 2940 ctcgtcgtgc tcacgaaccgtcatgacctg ggggcctggg gggactacag cctggggccc 3000 ctgggtcagc cgacggtggttccggactcc gtggacctgt ctccccaggg gctggccgct 3060 acgctgagca tggactggctattaataaac gagctcctgc aggtcaccga cgg 3113 <210> SEQ ID NO 53 <211>LENGTH: 761 <212> TYPE: DNA <213> ORGANISM: HSV-2 <400> SEQUENCE: 53gcgcccgctc gcggctcagc gcgaggccgc cggggtttac gacgcggtgc ggacctgggg 60gccagacgcg gaggccgaac cggaccagat ggaaaacacg tatctgctgc ccgacgatga 120cgccgccatg cccgcgggcg tcgggcttgg cgccaccccc gccgccgaca ccaccgccgc 180cgcctggccg gccgaaagcc acgccccccg cgccccctcc gaggacgcag attccattta 240cgagtcggtg agcgaggatg gggggcgcgt ctacgaggag atcccytggg ttcgggtata 300cgaaaacatc tgccttcgcc ggcaagacgc cggcggggcg gccccgccgg gagacgcccc 360ggactccccg tacatcgagg cggaaaatcc cctgtacgac tggggcgggt ctgccctctt 420ctcccctccg ggggccacac gcgccccgga cccgggacta agcctgtcgc ccatgcccgc 480ccgcccccgg accaacgcgc tggccaacga cggcccgaca aacgtcgccg ccctcagcgc 540cctgttgacg aagctcaaac gcggccgaca ccagagccat taaaaaaatg cgaccgccgg 600ccccaccgtc tcggtttccg gcccctttcc ccgtatgtct gttttcaata aaaagtaaca 660aacagagaaa aaaaaacagc gagttccgca tggtttgtcg tacgcaatta gctgtttatt 720gttttttttt tggggggggg aagagaaaaa gaaaaaagga g 761 <210> SEQ ID NO 54<211> LENGTH: 1037 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE:54 Met Ser Asp Ser Ala Leu Gln Val Pro Ala Pro Ala Gly Met Thr Pro 5 1015 Pro Ser Ala Pro Pro Pro Asn Gly Pro Leu Gln Val Leu Leu Gly Ser 20 2530 Leu Thr Asn Leu Arg Arg Pro Pro Ser Pro Ser Ser Glu Pro Ala Gly 35 4045 Ser Ala Asp Glu Pro Ala Phe Leu Ser Ala Ala Lys Leu Arg Ala Ala 50 5560 Thr Ala Ala Phe Leu Leu Ser Gly Ala Ala Val Gly Pro Ala Glu Ala 65 7075 80 Arg Ala Cys Trp His Pro Leu Leu Glu Gln Leu Cys Ala Leu His Arg 8590 95 Ala His Gly Leu Pro Glu Thr Ala Leu Leu Ala Glu Asn Leu Pro Gly100 105 110 Leu Leu Val His Arg Met Ala Val Ala Leu Pro Glu Thr Pro GluAla 115 120 125 Ala Phe Arg Glu Met Asp Val Ile Lys Asp Thr Val Leu AlaIle Thr 130 135 140 Gly Ser Asp Thr Thr His Ala Leu Glu Ala Ala Gly LeuArg Thr Thr 145 150 155 160 Ala Ala Leu Gly Pro Val Arg Val Arg Gln CysAla Val Glu Trp Ile 165 170 175 Asp Arg Trp Arg Thr Val Thr Gln Ser CysLeu Ala Met Asn Pro Arg 180 185 190 Thr Ser Leu Glu Ala Leu Gly Glu MetSer Leu Lys Met Ser Pro Val 195 200 205 Pro Leu Gly Gln Pro Gly Ala AsnLeu Thr Thr Pro Ala Tyr Ser Leu 210 215 220 Leu Phe Pro Ser Pro Ile ValGln Glu Gly Leu Arg Phe Leu Ala Leu 225 230 235 240 Val Ser Asn Trp ValThr Leu Phe Ser Ala His Leu Gln Arg Ile Asp 245 250 255 Asp Ala Ala LeuThr Pro Leu Thr Arg Ala Leu Phe Thr Leu Ala Leu 260 265 270 Val Asp GluTyr Leu Thr Thr Pro Asp Arg Gly Ala Val Val Pro Pro 275 280 285 Pro LeuLeu Ala Gln Phe Gln His Thr Val Arg Glu Ile Asp Pro Ala 290 295 300 IleMet Ile Pro Pro Leu Glu Ala Thr Lys Met Val Arg Ser Arg Glu 305 310 315320 Glu Val Arg Val Ser Thr Ala Leu Ser Arg Val Ser Pro Arg Ser Ala 325330 335 Cys Ala Pro Pro Gly Thr Leu Met Ala Arg Val Arg Thr Asp Ala Ala340 345 350 Val Phe Asp Pro Asp Val Pro Phe Leu Ser Ala Ser Ala Leu AlaIle 355 360 365 Phe Arg Pro Ala Val Thr Gly Leu Leu Gln Leu Gly Glu ProPro Ser 370 375 380 Ala Gly Ala Gln Gln Arg Leu Leu Ala Leu Leu Gln GlnThr Trp Ala 385 390 395 400 Leu Val Gln Asn Ser Asn Ser Pro Ser Val ValIle Asn Thr Leu Thr 405 410 415 Asp Ala Gly Phe Thr Pro Ala His Cys ThrGln Tyr Ile Ser Ala Leu 420 425 430 Glu Gly Phe Leu Val Ala Gly Val ProAla Arg Thr Pro Pro Gly His 435 440 445 Gly Leu Ser Glu Ile Gln Gln LeuPhe Gly Cys Ile Ala Leu Ala Gly 450 455 460 Ala Asn Val Phe Gly Leu AlaArg Glu Tyr Gly His Tyr Ala Gly Tyr 465 470 475 480 Val Lys Thr Phe ArgArg Ile Gln Gly Ala Ser Glu His Thr His Gly 485 490 495 Arg Leu Cys GluAla Val Gly Leu Ser Gly Gly Val Leu Ser Gln Thr 500 505 510 Leu Ala ArgIle Met Gly Pro Ala Val Pro Thr Glu His Leu Ala Ser 515 520 525 Leu ArgArg Thr Leu Val Gly Glu Phe Glu Thr Ala Glu Arg Arg Phe 530 535 540 SerAla Gly Gln Pro Ser Leu Leu Arg Glu Thr Ala Leu Ile Trp Leu 545 550 555560 Asp Val Tyr Gly Gln Thr His Trp Asp Leu Thr Pro Thr Thr Pro Ala 565570 575 Thr Pro Leu Ser Ala Leu Leu Pro Val Gly Pro Pro Ser His Ala Pro580 585 590 Ser Val His Leu Ala Ala Ala Thr Lys Ile Arg Phe Pro Ala LeuGlu 595 600 605 Gly Ile His Pro Asn Val Leu Ala Asp Pro Gly Phe Val ProTyr Val 610 615 620 Leu Ala Leu Val Val Gly Asp Ala Leu Arg Ala Thr CysAsn Ala Ala 625 630 635 640 Tyr Leu Pro Arg Pro Ile Glu Phe Ala Leu ArgVal Leu Ala Trp Ala 645 650 655 Arg Asp Phe Gly Leu Gly Tyr Leu Pro ThrVal Glu Gly His Arg Thr 660 665 670 Lys Leu Gly Ala Leu Ile Thr Leu LeuGlu Pro Ala Thr Arg Ala Gly 675 680 685 Val Gly Pro Thr Met Gln Met AlaAsp Asn Ile Glu Gln Leu Leu Arg 690 695 700 Glu Leu Tyr Val Ile Ala ArgGly Ala Val Glu Gln Leu Arg Pro Ala 705 710 715 720 Val Gln Leu Pro ProPro Gln Pro Pro Glu Val Gly Ser Ser Leu Leu 725 730 735 Leu Ile Ser MetTyr Ala Leu Ala Ala Arg Gly Val Leu Gln Glu Leu 740 745 750 Ala Glu ArgAla Asp Pro Leu Val Arg Gln Leu Glu Asp Ala Ile Val 755 760 765 Leu LeuArg Leu His Met Arg Thr Leu Ala Ala Phe Phe Glu Cys Arg 770 775 780 PheGlu Ser Asp Gly His Arg Leu Tyr Ala Val Val Ala Asp Ala His 785 790 795800 Glu Arg Leu Gly Pro Trp Arg Pro Glu Ala Met Gly Asp Ala Val Ser 805810 815 Gln Tyr Cys Gly Met Tyr His Asp Ala Lys Arg Ala Leu Val Ala Ser820 825 830 Leu Ala Gly Leu Arg Ser Val Val Thr Glu Thr Thr Ala His LeuGly 835 840 845 Val Cys Asp Glu Leu Ala Ala Gln Val Ser His Glu Gly AsnVal Leu 850 855 860 Ala Val Val Arg Arg Glu Ile His Gly Phe Leu Ala IleVal Ser Gly 865 870 875 880 Ile His Ala Arg Ala Ser Lys Leu Met Ser GlyAsp Gln Val Pro Gly 885 890 895 Phe Cys Tyr Met Ser Gln Phe Leu Ala ArgTrp Arg Arg Leu Ser Ala 900 905 910 Gly Tyr Gln Ala Ala Arg Ala Ala ThrGly Pro Glu Arg Val Ala Glu 915 920 925 Phe Val Gln Glu Leu His Asp ThrTrp Lys Gly Leu Gln Thr Glu Arg 930 935 940 Ala Leu Val Val Ala Pro PheAla Ser Ser Ala Asp Gln Arg Thr Ala 945 950 955 960 Ala Ile Gln Glu ValMet Ala His Ala Thr Glu Asp Ala Pro Pro Ser 965 970 975 Pro Ala Ala AspLeu Val Val Leu Thr Asn Arg His Asp Leu Gly Ala 980 985 990 Trp Gly AspTyr Ser Leu Gly Pro Leu Gly Gln Pro Thr Val Val Pro 995 1000 1005 AspSer Val Asp Leu Ser Pro Gln Gly Leu Ala Ala Thr Leu Ser Met 1010 10151020 Asp Trp Leu Leu Ile Asn Glu Leu Leu Gln Val Thr Asp 1025 1030 1035<210> SEQ ID NO 55 <211> LENGTH: 193 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 55 Arg Pro Leu Ala Ala Gln Arg Glu Ala Ala Gly ValTyr Asp Ala Val 5 10 15 Arg Thr Trp Gly Pro Asp Ala Glu Ala Glu Pro AspGln Met Glu Asn 20 25 30 Thr Tyr Leu Leu Pro Asp Asp Asp Ala Ala Met ProAla Gly Val Gly 35 40 45 Leu Gly Ala Thr Pro Ala Ala Asp Thr Thr Ala AlaAla Trp Pro Ala 50 55 60 Glu Ser His Ala Pro Arg Ala Pro Ser Glu Asp AlaAsp Ser Ile Tyr 65 70 75 80 Glu Ser Val Ser Glu Asp Gly Gly Arg Val TyrGlu Glu Ile Pro Trp 85 90 95 Val Arg Val Tyr Glu Asn Ile Cys Leu Arg ArgGln Asp Ala Gly Gly 100 105 110 Ala Ala Pro Pro Gly Asp Ala Pro Asp SerPro Tyr Ile Glu Ala Glu 115 120 125 Asn Pro Leu Tyr Asp Trp Gly Gly SerAla Leu Phe Ser Pro Pro Gly 130 135 140 Ala Thr Arg Ala Pro Asp Pro GlyLeu Ser Leu Ser Pro Met Pro Ala 145 150 155 160 Arg Pro Arg Thr Asn AlaLeu Ala Asn Asp Gly Pro Thr Asn Val Ala 165 170 175 Ala Leu Ser Ala LeuLeu Thr Lys Leu Lys Arg Gly Arg His Gln Ser 180 185 190 His <210> SEQ IDNO 56 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400>SEQUENCE: 56 Ser Pro Asn Thr Asp Val Arg Met Tyr Ser Gly Lys Arg Asn Gly5 10 15 <210> SEQ ID NO 57 <211> LENGTH: 15 <212> TYPE: PRT <213>ORGANISM: HSV-2 <400> SEQUENCE: 57 Tyr Leu Ala Ala Pro Thr Gly Ile ProPro Ala Phe Phe Pro Ile 5 10 15 <210> SEQ ID NO 58 <211> LENGTH: 15<212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 58 Gly Val Ala AlaAla Thr Pro Arg Pro Asp Pro Glu Asp Gly Ala 5 10 15 <210> SEQ ID NO 59<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE:59 Glu Glu Ile Pro Trp Val Arg Val Tyr Glu Asn Ile Cys Pro Arg 5 10 15<210> SEQ ID NO 60 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 60 Pro Gly Asp Ala Pro Asp Ser Pro Tyr Ile Glu AlaGlu Asn Pro 5 10 15 <210> SEQ ID NO 61 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: HSV-2 <400> SEQUENCE: 61 Pro Asp Ser Pro Tyr Ile Glu AlaGlu Asn Pro Leu Tyr Asp Trp 5 10 15 <210> SEQ ID NO 62 <211> LENGTH: 15<212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE: 62 Glu Asn Pro LeuTyr Asp Trp Gly Gly Ser Ala Leu Phe Ser Pro 5 10 15 <210> SEQ ID NO 63<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: HSV-2 <400> SEQUENCE:63 Ala Ile Asp Tyr Val His Cys Glu Gly Ile Ile His Arg Asp Ile 5 10 15<210> SEQ ID NO 64 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:HSV-2 <400> SEQUENCE: 64 Ala Phe Pro Val Ala Leu His Ala Val Asp Ala ProSer Gln Phe 5 10 15

What is claimed:
 1. An isolated polypeptide comprising at least an immunogenic portion of an HSV antigen, wherein said antigen comprises an amino acid sequence set forth in any one of SEQ ID NO: 2-3, 5-7, 10-12, 14-15, 17-18, 20-23, 25-26, 39-41, 45-47, 50-51, and 54-55.
 2. An isolated polynucleotide encoding a polypeptide of claim
 1. 3. An isolated polynucleotide of claim 2, wherein said polynucleotide comprises a sequence set forth in any one of SEQ ID NO: 1, 4, 8-9, 13, 16, 19,24, 34-38, 48-49, and 52-53.
 4. An isolated polypeptide comprising at least an immunogenic portion of a HSV UL46 antigen, wherein said antigen comprises an amino acid sequence set forth in SEQ ID NO: 15, and wherein said immunogenic portion is selected from the group consisting of SEQ ID NO: 27-33 and 59-62.
 5. An isolated polypeptide comprising at least an immunogenic portion of a HSV UL 1 5 antigen, wherein said antigen comprises an amino acid sequence set forth in SEQ ID NO: 26, and wherein said immunogenic portion is selected from the group consisting of SEQ ID NO: 56-57.
 6. An isolated polypeptide comprising at least an immunogenic portion of a HSV US3 antigen, wherein said antigen comprises an amino acid sequence set forth in SEQ ID NO: 12, and wherein said immunogenic portion comprises SEQ ID NO:
 63. 7. An isolated polypeptide comprising at least an immunogenic portion of a HSV US8A antigen, wherein said antigen comprises an amino acid sequence set forth in SEQ ID NO: 7, and wherein said immunogenic portion comprises SEQ ID NO:
 64. 8. A fusion protein comprising a polypeptide according to claim 1 and a fusion partner.
 9. A fusion protein according to claim 8, wherein the fusion partner comprises an expression enhancer that increases expression of the fusion protein in a host cell transfected with a polynucleotide encoding the fusion protein.
 10. A fusion protein according to claim 8, wherein the fusion partner comprises a T helper epitope that is not present within the polypeptide of claim
 1. 11. A fusion protein according to claim 8, wherein the fusion partner comprises an affinity tag.
 12. An isolated polynucleotide encoding a fusion protein according to claim
 8. 13. An isolated monoclonal or polyclonal antibody, or antigen-binding fragment thereof, that specifically binds to a polypeptide of claim
 1. 14. A pharmaceutical composition comprising a polypeptide according to claim 1 or a polynucleotide encoding said polypeptide, and a physiologically acceptable carrier.
 15. A pharmaceutical composition comprising a polypeptide according to claim 1, or a polynucleotide encoding said polypeptide, and an immunostimulant.
 16. The pharmaceutical composition of claim 15, wherein the immunostimulant is selected from the group consisting of a monophosphoryl lipid A, aminoalkyl glucosaminide phosphate, saponin, or a combination thereof.
 17. A method for stimulating an immune response in a patient, comprising administering to a patient a pharmaceutical composition according to any one of claims 14-16.
 18. A method for detecting HSV infection in a patient, comprising: (a) obtaining a biological sample from the patient; (b) contacting the sample with a polypeptide according to claim 1; and (c) detecting the presence of antibodies that bind to the polypeptide.
 19. The method according to claim 18, wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, cerebrospinal fluid and urine.
 20. A method for detecting HSV infection in a biological sample, comprising: (a) contacting the biological sample with a binding agent which is capable of binding to a polypeptide according to claim 1; and (b) detecting in the sample a polypeptide that binds to the binding agent, thereby detecting HSV infection in the biological sample.
 21. The method of claim 20, wherein the binding agent is a monoclonal antibody.
 22. The method of claim 20, wherein the binding agent is a polyclonal antibody.
 23. The method of claim 20 wherein the biological sample is selected from the group consisting of whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine.
 24. A diagnostic kit comprising a component selected from the group consisting of: (a) a polypeptide according to claim 1; (b) a fusion protein according to claim 8; (c) at least one antibody, or antigen-binding fragment thereof, according to claim 13; and (d) a detection reagent.
 25. The kit according to claim 24, wherein the polypeptide is immobilized on a solid support.
 26. The kit according to claim 24, wherein the detection reagent comprises a reporter group conjugated to a binding agent.
 27. The kit of claim 26, wherein the binding agent is selected from the group consisting of anti-immunoglobulins, Protein G, Protein A and lectins.
 28. The kit of claim 26, wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.
 29. A method for treating HSV infection in a patient, comprising the steps of: (a) obtaining peripheral blood cells from the patient; (b) incubating the cells in the presence of at least one polypeptide according to claim 1, such that T cells proliferate; and (c) administering to the patient the proliferated T cells.
 30. The method of claim 29, wherein the step of incubating the T cells is repeated one or more times.
 31. The method of claim 29, wherein step (a) further comprises separating T cells from the peripheral blood cells, and the cells incubated in step (b) are the T cells.
 32. The method of claim 29, wherein step (a) further comprises separating CD4+ cells or CD8+ T cells from the peripheral blood cells, and the cells proliferated in step (b) are CD4+ or CD8+ T cells.
 33. The method of claim 29, wherein step (a) further comprises separating gamma/delta T lymphocytes from the peripheral blood cells, and the cells proliferated in step (b) are gamma/delta T lymphocytes.
 34. The method of claim 29, wherein step (b) further comprises cloning one or more T cells that proliferated in the presence of the polypeptide.
 35. A pharmaceutical composition for the treatment of HSV infection in a patient, comprising T cells proliferated in the presence of a polypeptide of claim 1, in combination with a physiologically acceptable carrier.
 36. A method for treating HSV infection in a patient, comprising the steps of: (a) incubating antigen presenting cells in the presence of at least one polypeptide of claim 1; (b) administering to the patient the incubated antigen presenting cells.
 37. The method of claim 36, wherein the antigen presenting cells are selected from the group consisting of dendritic cells. macrophage cells, B cells fibroblast cells, monocyte cells, and stem cells.
 38. A pharmaceutical composition for the treatment of HSV infection in a patient, comprising antigen presenting cells incubated in the presence of a polypeptide of claim 1, in combination with a physiologically acceptable carrier. 